Compositions and methods for biological production of butane-based compounds from a c1 substrate

ABSTRACT

The present disclosure relates to biosynthetic methods for producing propylene from C 1  substrates (e.g., methane, methanol, carbon monoxide, syngas) and to genetically engineered organisms having propylene biosynthesis capability, as well as engineered organisms having a butyrate/butanol-producing pathway.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200206_403C1_SEQUENCE_LISTING.txt. The text file is 363 KB, was created on Jun. 28, 2017, and is being submitted electronically via EFS-Web.

BACKGROUND

Propylene is primarily produced as a by-product of petroleum refining and of ethylene production using a steam cracking process. Propylene is separated from a mixture of hydrocarbons obtained from cracking or other refining processes by fractional distillation. Propylene is typically produced from non-renewable fossil fuels, petroleum, natural gas, and to a lesser extent coal. Propylene can also be produced in on-purpose reactions, such as propane dehydrogenation, metathesis or syngas-to-olefins plants.

Propylene is a major industrial chemical intermediate that is converted into a variety of chemicals and plastics. Manufacturers of polypropylene account for nearly two thirds of worldwide propylene demand. Polypropylene is a plastic that is used for the manufacture of films, packaging, caps, closures, and individual parts for the electrical and automotive industry. Propylene is also used to produce chemicals including acrylonitrile, oxo chemicals, propylene oxide, cumene, isopropanol, acrylic acid, butanol, and butanediol.

Currently, refinery by-product production of propylene can no longer satisfy market demand. There is a need for alternative processes for on-purpose propylene production, particularly a green process that exhibits increased safety, decreased harmful waste and emissions, and savings in cost and energy compared to petrochemically-derived propylene.

SUMMARY

In one aspect, the present disclosure provides non-naturally occurring C₁ metabolizing organisms, wherein the non-naturally occurring C₁ metabolizing organisms convert a C₁ substrate to propylene. In certain embodiments, the C₁ metabolizing organism is: a non-naturally occurring C₁ metabolizing bacterium; a non-naturally occurring obligate C₁ metabolizing organism; a non-naturally occurring C₁ metabolizing organism, wherein the non-naturally occurring C₁ metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; a non-naturally occurring syngas utilizing bacterium that naturally possesses the ability to utilize syngas; or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO. In certain aspects, the non-naturally occurring C₁ metabolizing organisms include an exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid (e.g., 4-oxalocrotonate decarboxylase). In further aspects, the non-naturally occurring C₁ metabolizing organisms do not have a functional PHB synthase or a substantial amount of functional PHB synthase.

In another aspect, non-naturally occurring C₁ metabolizing organisms include an exogenous nucleic acid encoding a crotonyl CoA thioesterase. The non-naturally occurring C₁ metabolizing organism may be a non-naturally occurring C₁ metabolizing bacterium; a non-naturally occurring obligate C₁ metabolizing organism; a non-naturally occurring C₁ metabolizing organism, wherein the C₁ metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; a non-naturally occurring syngas utilizing bacterium that naturally possesses the ability to utilize syngas;or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO.

In yet another aspect, non-naturally occurring C₁ metabolizing organisms include an exogenous nucleic acid encoding a crotonase. In certain embodiments, the non-naturally occurring C₁ metabolizing organism is: a non-naturally occurring C₁ metabolizing bacterium; a non-naturally occurring obligate C₁ metabolizing organism; a non-naturally occurring C₁ metabolizing organism, wherein the organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; a non-naturally occurring syngas utilizing bacterium that naturally possesses the ability to utilize syngas; or a non-naturally occurring CO utilizing bacterium, wherein the CO utilizing bacterium naturally possesses the ability to utilize CO.

In another aspect, the present disclosure provides non-naturally occurring microbial organisms, wherein the non-naturally occurring microbial organisms include an exogenous nucleic acid encoding 4-oxalocrotonate decarboxylase, and wherein the non-naturally occurring microbial organisms convert a carbon substrate to propylene. In certain embodiments, the non-naturally occurring microbial organisms further include an exogenous nucleic acid encoding crotonase, or an exogenous nucleic acid encoding crotonase and an exogenous nucleic acid encoding crotonyl thioesterase. In certain embodiments, the non-naturally occurring microbial organisms do not have a functional PHB synthase or a substantial amount of functional PHB synthase.

Also disclosed herein are methods of producing propylene in non-naturally occurring organisms described herein, wherein the methods comprise culturing the non-naturally occurring organisms under conditions sufficient to produce propylene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary pathway for synthesis of propylene from acetyl CoA in a genetically modified organism. Enzymes and genes encoding enzymes for transformation of the identified substrates to products include: phaA, phaB, crotonase, crotonyl CoA thioesterase, and 4-oxalocrotonate decarboxylase.

FIGS. 2A-D show exemplary 4-oxalocrotonate decarboxylase (4-OD) amino acid sequences that may be used in propylene synthesis pathways as described herein.

FIG. 3 shows exemplary crotonyl-CoA thioesterase amino acid sequences that may be used in propylene synthesis pathways as described herein.

FIGS. 4A-E show exemplary crotonase amino acid sequences that may be used in propylene synthesis pathways as described herein.

FIG. 5 shows SDS-PAGE analysis of heterogeneously expressed 4-OD genes in E. coli. The first lane for each sample shows total cell protein and the second lane for each sample shows soluble protein following lysis and clarification. The arrow on the left shows the approximate migration of 4-OD proteins (note sequence variation causes slight changes in migration for each individual 4-OD sequence).

FIG. 6 shows sample 4-OD activity assays run under conditions as described showing decarboxylation of 4-oxalocrotonate.

FIG. 7 shows a sample chromatogram of propylene detected on a HP5890 GC-FID under the assay conditions as described.

DETAILED DESCRIPTION

The instant disclosure provides compositions and methods for the biocatalysis of propylene and other desirable intermediates or products from methane or other C₁ substrates using genetically engineered C₁ metabolizing organisms. The instant disclosure also provides compositions and methods for the biocatalysis of propylene from carbon substrates using novel metabolic enzyme(s) in genetically engineered microbial organisms.

Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

In the present description, the term “about” means ±20% of the indicated range, value, or structure, unless otherwise indicated. The term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include” and “have” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting. The term “comprise” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

As used herein, the term “non-naturally occurring” when used in reference to a bacterium or an organism means that the bacterium or organism has at least one genetic alternation that is not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruption of the bacterium or organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous or homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary enzymes include enzymes within a crotonate synthesis pathway or propylene synthesis pathway. Genetic modifications to nucleic acid molecules encoding enzymes, or functional fragments thereof, can confer a biochemical reaction capability or a metabolic pathway capability to the non-naturally occurring organism that is altered from its naturally occurring state.

As used herein, the term “host” refers to a bacterium or organism that has not yet been genetically modified with the capability to convert a carbon substrate to crotonyl-CoA, crotonic acid, or propylene, as disclosed herein. A host C₁ metabolizing bacterium or organism is selected for transformation with at least one exogenous nucleic acid encoding an enzyme to yield a non-naturally occurring C₁ metabolizing bacterium or organism with the capability to convert a C₁ substrate to crotonyl-CoA, crotonic acid, or propylene. A host microbial organism is selected for transformation with at least one exogenous nucleic acid encoding 4-oxalocrotonate decarboxylase to yield a non-naturally occurring microbial organism with the capability to convert a carbon substrate to propylene. A host bacterium or organism may already possess other genetic modifications conferring it with desired properties, unrelated to propylene synthesis pathways and intermediates disclosed herein. For example, a host bacterium or organism may possess genetic modifications conferring high growth, tolerance of contaminants or particular culture conditions, or ability to metabolize different carbon substrates.

As used herein, the term “microbial organism,” “microorganism”, “microbes”, or “organism” refers to any prokaryotic or eukaryotic microbial species from the domains of Archaea, Bacteria, or Eukarya. The term is intended to include prokaryotic or eukaryotic cells or organisms having microscopic size.

As used herein, the term “C₁ metabolizing bacterium” refers to any bacterium that has the ability to oxidize a C₁ compound (i.e., does not contain carbon-carbon bonds). A C₁ metabolizing bacterium may or may not use other carbon substrates, such as sugars and complex carbohydrates, for energy and biomass.

As used herein, the term “obligate C₁ metabolizing organism” refers to those organisms which exclusively use organic compounds that do not contain carbon-carbon bonds (C₁ substrate) for the generation of energy.

As used herein, the term “C₁ metabolizing organism” refers to any organism that has the ability to oxidize a C₁ substrate (i.e., does not contain carbon-carbon bonds) but may or may not use other carbon substrates, such as sugars and complex carbohydrates, for energy and biomass. A C₁ metabolizing organism includes bacteria, yeast (not including Pichia pastoris), and Archaea. A C₁ metabolizing organism includes C₁ metabolizing bacteria.

As used herein, the term “methanotrophic bacterium” refers to any methylotrophic bacterium that has the ability to oxidize methane as its primary carbon and energy source.

As used herein, the term “methylotrophic bacterium” refers to any bacterium capable of oxidizing organic compounds that do not contain carbon-carbon bonds. An “obligate methylotrophic bacterium” is a bacterium which is limited to the use of carbon substrates that do not contain carbon-carbon bonds for the generation of energy. Facultative methylotrophs are able to utilize multi-carbon compounds in addition to single carbon substrates.

As used herein, the term “CO utilizing bacterium” refers to a bacterium that naturally possesses the ability to oxidize carbon monoxide (CO) as a source of carbon and energy. Carbon monoxide may be utilized from “synthesis gas” or “syngas”, a mixture of carbon monoxide and hydrogen produced by gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Syngas may also include CO₂, methane, and other gases in smaller quantities. CO utilizing bacterium does not include bacteria that must be genetically modified for growth on CO as its carbon source.

As used herein, the term “C₁ substrate” or “C₁ compound” refers to any carbon containing molecule or composition that lacks a carbon-carbon bond. C₁ substrates include methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines (methylamine, dimethylamine, trimethylamine, etc.), methylated thiols, methyl halogens (bromomethane, chloromethane, iodomethane, dichloromethane, etc.), or cyanide.

As used herein, the term “propylene”, also known as 1-propene, propene, or methylethylene, refers to an unsaturated organic compound having the chemical formula C₃H₆ and molecular mass of 42.08 g/mol. It has one double bond and is the second simplest member of the alkene class of hydrocarbons. Propylene is a gas at room temperature and atmospheric pressure. Propylene is a structural isomer of cyclopropane.

As used herein, “exogenous” means that the referenced molecule (e.g., nucleic acid) or referenced activity (e.g., enzyme activity) is introduced into the host bacterium or organism. The molecule can be introduced, for example, by introduction of a nucleic acid into the host genetic material such as by integration into a host chromosome or by introduction of a nucleic acid as non-chromosomal genetic material, such as on a plasmid. When the term is used in reference to expression of an encoding nucleic acid, it refers to introduction of the encoding nucleic acid in an expressible form into the bacterium or organism. When used in reference to an enzymatic activity, the term refers to an activity that is introduced into the host reference bacterium or organism. Therefore, the term “endogenous” or “native” refers to a referenced molecule or activity that is present in the host bacterium or organism. The term “heterologous” refers to a molecule or activity that is derived from a source other than the referenced species or strain whereas “homologous” refers to a molecule or activity derived from the host bacterium or organism. Accordingly, a bacterium or organism comprising an exogenous nucleic acid of the invention can utilize either or both a heterologous or homologous nucleic acid.

It is understood that when more than one exogenous nucleic acid is included in a bacterium or organism that the more than one exogenous nucleic acid refers to the referenced encoding nucleic acid or enzymatic activity, as discussed above. It is also understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host bacterium or organism on separate nucleic acid molecules, on a polycistronic nucleic acid molecule, on a single nucleic acid molecule encoding a fusion protein, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein, an organism can be modified to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein (e.g., propylene pathway enzyme or protein). Where two exogenous nucleic acids encoding propylene synthesis activity are introduced into a host organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid molecule, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or enzymatic activities refers to the number of encoding nucleic acids or the number of enzymatic activities, not the number of separate nucleic acid molecules introduced into the host organism.

As used herein, “nucleic acid” refers to a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acids include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), both of which may be single or double stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.

As used herein, “crotonic acid” or “trans-2-butenoic acid” refers to a short-chain unsaturated carboxylic acid with the formula CH₃CH═CHCO₂H. As used herein, crotonic acid includes “crotonate”, a salt or ester of crotonic acid.

As used herein, “4-oxalocrotonate” refers to an unsaturated carboxylic acid with the formula HO₂C(C═O)CH₂CH═CHCO₂— (keto form) or HO₂C(COH)═CHCH═CHCO₂— (enol form), which interconvert spontaneously. As used herein, 4-oxalocrotonate also includes salts or esters, the equivalent molecule with the alternate acid group deprotonated, the doubly unprotonated basic form of the molecule, or the doubly protonated acid form of the molecule.

As used herein, “4-oxalocrotonate decarboxylase” or “4-OD” or “4-oxalocrotonate carboxy-lase (2-oxopent-4-enoate-forming)” refers to an enzyme family that catalyzes the decarboxylation of 4-oxalocrotonate to 2-oxopent-4-enoate and CO₂. 4-OD is involved in the meta-cleavage pathway for the degradation of phenols, modified phenols, and catechols. As disclosed herein, 4-OD is used to catalyze the decarboxylation of crotonic acid to propylene and CO₂. As used herein, 4-OD also encompasses mutants or variants of a native 4-OD enzyme that has reduced or eliminated decarboxylation activity on 4-oxalocrotonate, but retains or has increased decarboxylation activity on crotonic acid as a substrate. 4-OD also refers to any enzyme capable of catalyzing the decarboxylation of crotonic acid to propylene and CO₂.

As used herein, “crotonyl CoA thioesterase” refers to an enzyme that catalyzes the conversion of crotonyl-CoA to crotonic acid.

As used herein, “crotonase” or “3-hydroxybutyryl-CoA dehydratase” or “enoyl-CoA hydratase” refers to an enzyme involved in the butyrate/butanol-producing pathway, which catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA.

As used herein, “β-ketothiolase” refers to an enzyme in the PHB synthesis pathway that catalyzes the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. Biosynthesis of PHB in most bacteria is initiated by β-ketothiolase.

As used herein, “acetoacetyl coenzyme A reductase” refers to an enzyme in the PHB synthesis pathway that catalyzes the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA.

As used herein, “PHB synthase”, also known as “PHB polymerase”, refers to an enzyme in the PHB synthesis pathway that converts a hydroxybutyryl-CoA monomer into polyhydroxybutyrate.

As used herein, “polyhydroxybutyrate” or “PHB” refers to a homopolymer of hydroxybutyric acid units. PHB is a type of polyhydroxyalkanoate (PHA), which is a biological polyester. Polyhydroxybutyrate is a crystalline thermoplastic synthesized by a broad range of bacteria as a form of energy storage molecule. Poly-3-hydroxybutyrate (P3HB) form is the most common type of PHB, but other PHBs include poly-4-hydroxybutytrate (P4HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-co-4HB), or other copolymers.

C₁ Metabolizing Bacteria or Organisms

A variety of C₁ metabolizing host organisms can be transformed or genetically engineered to produce a product of interest (e.g., propylene, crotonic acid, or crotonyl-CoA).

In certain embodiments, a C₁ metabolizing organism may be a C₁ metabolizing bacterium, which refers to any bacterium that has the ability to oxidize a C₁ compound. A C₁ metabolizing bacterium may also use other carbon substrates, such as sugars and complex carbohydrates, for energy and biomass. A C₁ metabolizing bacterium includes methanotrophic bacteria (methylotrophic bacterium that has the ability to oxidize methane as an energy source) or methylotrophic bacteria (any bacterium capable of oxidizing organic compounds that do not contain carbon-carbon bonds). Methanotrophic bacteria are classified into three groups based on their carbon assimilation pathways and internal membrane structure: type I (gamma proteobacteria), type II (alpha proteobacteria, and type X (gamma proteobacteria). Type I methanotrophs use the ribulose monophosphate (RuMP) pathway for carbon assimilation whereas type II methanotrophs use the serine pathway. Type X methanotrophs use the RuMP pathway but also express low levels of enzymes of the serine pathway. Methanotrophic bacteria are grouped into several genera: Methylomonas, Methylobacter, Methylococcus, Methylocystis, Methylosinus, Methylomicrobium, Methanomonas, and Methylocella. Exemplary methantrophic bacteria include: Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402), Methylosinus trichosporium OB3b (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris, Methylocella palustris (ATCC 700799), Methylocella tundrae, Methylacidiphilum infernorum, Methylibium petroleiphilum, and Methylomicrobium alcaliphilum. Methylotrophic bacteria encompass a diverse group, including both gram-negative and gram-positive genera. Methylotrophic bacteria include facultative methylotrophs (have the ability to oxidize organic compounds which do not contain carbon-carbon bonds, but may also utilize other carbon substrates such as sugars and complex carbohydrates), obligate methylotrophs (limited to the use of organic compounds that do not contain carbon-carbon bonds), and methanotrophic bacteria. Examples of methylotrophic genera include: Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, and Pseudomonas. Exemplary methylotrophic bacteria include: Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylobacterium nodulans, Methylomonas clara, Methylibium petroleiphilum, Methylobacillus flagellates, Silicibacter pomeroyi DSS-3, Burkholderia phymatum STM815, Granulibacter bethesdensis NIH1.1, and Paracoccus denitrificans.

A C₁ metabolizing bacterium includes bacteria that utilize formate or cyanide or naturally possesses the ability to utilize syngas or carbon monoxide (CO) (Wu et al., PLoS Genet. 1:e65, 2005; Abrini et al., Arch. Microbiol. 161:345, 1994; WO 2008/028055; Tanner et al., Int. J. Syst. Bacteriol. 43:232-236, 1993). Exemplary bacteria that naturally possesses the ability to utilize CO or syngas include Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium woodii, Clostridium neopropanologen, Pseudomonas carboxidovorans, Rhodospirillum rubrum, Thermincola carboxydiphila, Thermincola potens, Thermoanaerobacter thermohydrosulfuricus, Ralstonia eutropha, and Eurobacterium limosum. A C₁ metabolizing bacterium also includes bacteria that can cleave methyl groups from organic compounds, including choline (de Vries et al., FEMS Microbiol. Rev. 6:57-101, 1990) or the pesticide carbofuran (Topp et al., Appl. Environ. Microbiol. 59:3339-3349, 1993), and utilize them as a sole source of carbon.

C₁ metabolizing organisms include bacteria, yeast (not including Pichia pastoris), and Archaea. A C₁ metabolizing organism may be obligate or facultative. Examples of C₁ metabolizing organisms include: Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, Pseudomonas, Candida, Yarrowia, Hansenula, Pichia (not including Pichia pastoris), Torulopsis, and Rhodotorula. Additional examples of C₁ (carbon monoxide) metabolizing organisms include: Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium Woodii, and Clostridium neopropanologen.

A variety of C₁ substrates may be used by the C₁ metabolizing organisms. It is understood to one of skill in the art that selection of a C₁ substrate may be determined by which host organism is selected. For example, obligate methanotrophic bacteria are limited to the use of methane as a carbon source, whereas some methylotrophic bacteria may use a variety of C₁ compounds, such as methane, methanol, methylated amines, halomethanes, and methylated compounds containing sulfur (reviewed in Hanson and Hanson, 1996, Microbiological Rev. 60:439-471). Non-limiting examples of C₁ substrates include: methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines (methylamine, dimethylamine, trimethylamine, etc.), methylated thiols, or methyl halogens (bromomethane, chloromethane, iodomethane, dichloromethane, etc.). Some facultative methanotrophs and facultative methylotrophs can also grow on multi-carbon compounds. A C₁ metabolizing organism may also be adapted or genetically modified to use a different C₁ substrate in addition to, or instead of its usual C₁ substrate. Alternatively, a C₁ metabolizing organism may be adapted or genetically modified to use a multi-carbon substrate in addition to its usual C₁ substrate. A selected C₁ metabolizing organism may also undergo strain adaptation under selective conditions to identify variants with improved properties for production. Improved properties may include increased growth rate, yield of desired products (e.g., propylene), and tolerance of likely process contaminants. In a particular embodiment, a high growth variant C₁ metabolizing organism, which is an organism capable of growth on a C₁ substrate as the sole carbon and energy source and which possesses an exponential phase growth rate that is faster (i.e., shorter doubling time) than its parent, reference, or wild-type organism, is selected ((see, e.g., U.S. Pat. No. 6,689,601).

Each of the organisms of this disclosure may be grown as an isolated culture, with a heterologous organism that may aid in growth, or one or more of C₁ metabolizing bacteria may be combined to generate a mixed culture.

Microbial Organisms

A variety of microbial host organisms can be transformed or genetically engineered to include a novel metabolic pathway and associated enzymes as described herein to produce propylene from a carbon substrate. Exemplary bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi species include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Rhizopus arrhizus, Rhizobus oryzae, and Yarrowia lipolytica. It is understood that any suitable microbial host organism can be used to introduce suitable genetic modifications (e.g., nucleic acid encoding 4-OD) to produce propylene.

Non-Naturally Occurring Organisms

Non-naturally occurring organisms as described herein can be produced by introducing expressible nucleic acid(s) encoding one or more enzymes involved in a desired biosynthetic pathway (e.g., propylene synthesis). Depending on the host organism selected for biosynthesis, nucleic acid(s) for one, some, or all of a particular biosynthetic pathway can be expressed. For example, if a selected host is deficient in one or more enzymes for a desired biosynthetic pathway (e.g., propylene synthesis), then expressible nucleic acid(s) for the deficient enzyme(s) are introduced into the host for subsequent exogenous expression. Alternatively, if a selected host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) to achieve the desired biosynthesis. Thus, non-naturally occurring organisms of the invention can be produced by introduced exogenous enzyme activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme activities that, together with one or more endogenous enzymes, produces the desired product, such as propylene. In some embodiments, non-naturally occurring organisms as described herein can also include other genetic modifications that facilitate or optimize a desired biosynthetic pathway or that confer other useful functions onto the host. For example, if a selected host exhibits endogenous expression of an enzyme that inhibits propylene biosynthetic pathway, then the host may be genetically modified so that it does not produce a functional enzyme or a substantial amount of a functional enzyme.

In certain embodiments, the present disclosure provides a non-naturally occurring C₁ metabolizing bacterium; a non-naturally occurring obligate C₁ metabolizing organism; a non-naturally occurring C₁ metabolizing organism, wherein the non-naturally occurring C₁ metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO, wherein the non-naturally occurring C₁ metabolizing bacterium or organism converts a C₁ substrate to propylene.

In certain embodiments, the present disclosure provides a non-naturally occurring C₁ metabolizing organism that is not Pichia pastoris and is capable of converting a C₁ substrate into propylene. In certain embodiments, the non-naturally occurring C₁ metabolizing organism is not Pichia pastoris and is a C₁ metabolizing bacterium capable of converting a C₁ substrate into propylene.

In certain embodiments, the non-naturally occurring C₁ metabolizing organism is an obligate C₁ metabolizing organism capable of converting a C₁ substrate into propylene. In certain embodiments, the obligate C₁ metabolizing organism is a C₁ metabolizing bacterium, such as a methanotrophic or methylotrophic bacterium capable of converting a C₁ substrate into propylene. In further embodiments, a C₁ metabolizing bacterium may be a syngas utilizing bacterium that naturally possesses the ability to use syngas and convert a C₁ substrate in the syngas into propylene. In still further embodiments, a C₁ metabolizing bacterium is a CO utilizing bacterium that naturally possesses the ability to use CO and convert the CO into propylene.

In certain embodiments, any of the non-naturally occurring C₁ metabolizing organisms as described herein includes an exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid. The exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid is expressed in a sufficient amount to produce propylene.

In further embodiments, the exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid encodes 4-oxalocrotonate decarboxylase (4-OD). 4-OD is an enzyme that catalyzes the decarboxylation of 4-oxalocrotonate to 2-oxopent-4-enoate and CO₂. 4-OD is involved in the meta-cleavage pathway for the degradation of phenols, modified phenols, and catechols. As disclosed herein, an exogenous nucleic acid encoding 4-OD is used to genetically engineer a novel propylene biosynthesis pathway in a non-naturally occurring organisms; 4-OD is used to catalyze decarboxylation of crotonic acid to propylene and CO₂ (see FIG. 1). Sources of 4-OD encoding nucleic acid molecules may include any species, prokaryotic or eukaryotic, where the encoded gene product is capable of catalyzing decarboxylation of crotonic acid to propylene and CO₂. Exemplary amino acid sequences of 4-OD are shown in FIGS. 2A-D.

Non-naturally occurring C₁ metabolizing organisms that have an exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid (e.g., 4-OD) may also include one or more exogenous nucleic acids to confer a propylene biosynthetic pathway onto the C₁ metabolizing organism. For example, depending on which C₁ metabolizing host organism is selected, one or more exogenous nucleic acids may need to be introduced into the C₁ metabolizing organism along with 4-OD in order to provide a non-naturally occurring C₁ metabolizing organism with the ability to produce crotonic acid, the substrate for propylene conversion by 4-OD. In certain embodiments, a non-naturally occurring C₁ metabolizing organism that has an exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid (e.g., 4-OD) may further include an exogenous nucleic acid encoding crotonase and/or an exogenous nucleic acid encoding a crotonyl-CoA thioesterase. An exogenous nucleic acid encoding crotonase and an exogenous nucleic acid encoding crotonyl-CoA thioesterase are expressed in a sufficient amount to produce propylene. In a specific example, a type II methanotrophic bacterium, which possesses an endogenous PHB synthesis pathway and produces 3-hydyroxybutyryl-CoA, an intermediate in the PHB synthesis pathway, may comprise exogenous nucleic acids encoding a crotonase and/or a crotonyl-CoA thioesterase in addition to 4-OD (see FIG. 1). Crotonase and crotonyl-CoA thioesterase are provided to catalyze the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA and conversion of crotonyl-CoA to crotonic acid, respectively. It is understood that any combination of one or more enzymes that can be used to engineer a propylene biosynthesis pathway can be included in a non-naturally occurring C₁ metabolizing organism of the invention.

In certain embodiments, the present disclosure provides a non-naturally occurring C₁ metabolizing bacterium; a non-naturally occurring obligate C₁ metabolizing organism; a non-naturally occurring C₁ metabolizing organism, wherein the non-naturally occurring C₁ metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO, wherein the non-naturally occurring C₁ metabolizing bacterium or organism comprises an exogenous nucleic acid encoding a crotonyl-CoA thioesterase.

In certain embodiments, the present disclosure provides a non-naturally occurring C₁ metabolizing organism that is not Pichia pastoris and includes an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In further embodiments, a non-naturally occurring C₁ metabolizing organism that is not Pichia pastoris, may be an obligate C₁ metabolizing organism containing an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In still further embodiments, the C₁ metabolizing organism may be a C₁ metabolizing bacterium or an obligate C₁ metabolizing bacterium containing an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In some embodiments, a C₁ metabolizing bacterium is a methanotrophic or methylotrophic bacterium containing an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In certain embodiments, a C₁ metabolizing bacterium may be a syngas utilizing bacterium that naturally possesses the ability to use syngas and contains an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In some embodiments, a C₁ metabolizing bacterium may be a CO utilizing bacterium that naturally possesses the ability to use CO and contains an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase.

Crotonyl-CoA thioesterase refers to an enzyme that catalyzes the conversion of crotonyl-CoA to crotonic acid. Sources of crotonyl-CoA thioesterase encoding nucleic acids may include any species, prokaryotic or eukaryotic, where the encoded gene product is capable of catalyzing the conversion of crotonyl-CoA to crotonic acid. Exemplary amino acid sequences for crotonyl-CoA thioesterase are shown in FIG. 3. Crotonic acid is an intermediate in the engineered propylene biosynthetic pathway disclosed herein and is a useful product in itself for the preparation of polyvinyl acetate copolymers, a synthetic butyl rubber softener, a variety of resins, fungicides, pharmaceutical intermediates, plasticizers, cosmetic polymers for hair care, and adhesives. Reduction of crotonic acid yields crotonaldehyde. Key products made from crotonaldehyde are sorbic acid, potassium sorbate (a preservative), and trimethylhydroquinone (an intermediate used in Vitamin E manufacture).

In certain embodiments, the present disclosure provides a non-naturally occurring C₁ metabolizing bacterium; a non-naturally occurring obligate C₁ metabolizing organism; a non-naturally occurring C₁ metabolizing organism, wherein the non-naturally occurring C₁ metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO, wherein any of the aforementioned non-naturally occurring C₁ metabolizing bacterium or organism includes an exogenous nucleic acid encoding a crotonase.

In certain embodiments, the present disclosure provides a non-naturally occurring C₁ metabolizing organism that is not Pichia pastoris and includes an exogenous nucleic acid molecule encoding a crotonase. In certain embodiments, the non-naturally occurring C₁ metabolizing organism that is not Pichia pastoris is a C₁ metabolizing bacterium containing an exogenous nucleic acid molecule encoding a crotonase, such as a methanotrophic or methylotrophic bacterium. In some embodiments, a C₁ metabolizing bacterium is a syngas utilizing bacterium that naturally possesses the ability to use syngas and contains an exogenous nucleic acid molecule encoding a crotonase. In some embodiments, a C₁ metabolizing bacterium is a CO utilizing bacterium that naturally possesses the ability to use CO and contains an exogenous nucleic acid molecule encoding a crotonase. In further embodiments, a non-naturally occurring C₁ metabolizing organism is an obligate C₁ metabolizing organism containing an exogenous nucleic acid molecule encoding a crotonase, such as an obligate C₁ metabolizing bacterium.

Crotonase, also known as 3-hydroxybutyryl-CoA dehydratase, is an enzyme involved in the butyrate/butanol-producing pathway. Crotonase catalyzes the dehydration of 3-hydyroxybutyryl-CoA to crotonyl-CoA. 3-hydroxybutyryl-CoA can be an R or S stereoisomer. Enzymes that produce and convert 3-hydroxybutyryl-CoA have defined stereospecificity preferences. Sources of crotonase encoding nucleic acids may include any species, prokaryotic or eukaryotic, where the encoded gene product is capable of catalyzing dehydration of 3-hydyroxybutyryl-CoA to crotonyl-CoA. Exemplary amino acid sequences for crotonase are shown in FIGS. 4A-E. Crotonyl-CoA is a useful intermediate that can be a substrate for an engineered propylene biosynthetic pathway (via conversion to crotonic acid by crotonyl-CoA thioesterase, which is then converted to propylene by 4-OD) or an engineered butanol/butyraldehyde biosynthetic pathway (via conversion to butyryl-CoA by butyryl-CoA dehydrogenase, which is then converted to butanol or butyraldehyde by bi-functional aldehyde dehydrogenase) (see FIG. 1).

In certain embodiments, the invention provides for a non-naturally occurring C₁ metabolizing bacterium; a non-naturally occurring obligate C₁ metabolizing organism; a non-naturally occurring C₁ metabolizing organism, wherein the non-naturally occurring C₁ metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO; wherein the non-naturally occurring C₁ metabolizing bacterium or organism converts a C₁ substrate to propylene, and further wherein the non-naturally occurring C₁ metabolizing bacterium or organism does not have a functional PHB synthase or a substantial amount of a functional PHB synthase.

In certain embodiments, a non-naturally occurring C₁ metabolizing organism according to any of the embodiments disclosed herein is a C₁ metabolizing bacterium that does not have a functional PHB synthase or a substantial amount of functional PHB synthase.

PHB synthase is a polyhydroxybutyrate synthesis pathway enzyme that converts hydroxybutyryl-CoA monomers into polyhydroxybutyrate (PHB) (see FIG. 1). Many prokaryotes synthesize PHB as a carbon and energy storage material. Some yeast and other eukaryotic cells may contain small amounts of low molecular mass PHBs. Two PHB synthesis pathways are known in bacteria. In one pathway, PHB is synthesized from acetyl-CoA as a result of sequential action of three enzymes: β-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase (e.g., Methylobacterium extorquens, Methylosinus trichosporium OB3b, Methylocystis and Methylosinus species). PHB synthesis can also be synthesized by: β-ketothiolase, acetocetyl-CoA reductase, crotonyl-CoA hydratases (crotonases), and PHB synthase (e.g., Methylobacterium rhodesianum) (see, e.g., Mothes et al., Arch. Microbiol. 161:277-280, 1994; Mothes et al., Can. J. Microbiol. 41:68-72, 1995). Generally, Type II methanotrophs accumulate PHB, whereas type I methanotrophs do not. As used herein, “not having a functional PHB synthase” means that its gene expression or protein activity has been reduced to undetectable levels. Reducing gene expression or protein activity of PHB synthase may be accomplished by deletion of some or all of the gene's coding sequence, introduction of one or more nucleotides into the gene's open reading frame resulting in translation of a nonsense or non-functional protein product, expressing interfering RNA or antisense sequences that target the gene, or other methods known in the art. As used herein, “not having a substantial amount of a functional PHB synthase” means that an organism has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less gene expression or protein activity of PHB synthase as compared to a wild type organism that has a polyhydroxybutyrate synthesis pathway.

In a specific embodiment, PHB synthase is encoded by phaC or phbC. Exemplary PHB synthase amino acid sequences are provided in Table 1. In another specific embodiment, a non-naturally occurring C₁ metabolizing organism that is capable of converting a C₁ substrate to propylene as disclosed herein does not produce a substantial amount of polyhydroxybutryate. As used herein, “not producing a substantial amount of polyhydroxybutyrate” means that an organism produces at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less polyhydroxybutyrate as compared to a wildtype organism that has a polyhydroxybutyrate synthesis pathway. By knocking out PHB function in C₁ metabolizing organisms with an endogenous PHB synthesis pathway through genetic modification (e.g., allelic exchange of phaC or phbC with non-functional gene), PHB production may be inhibited, allowing more 3-hydroxybutyryl-CoA to be funneled into conversion to crotonyl-CoA by crotonase (see FIG. 1). Alternatively, a C₁ metabolizing organism, which is not naturally capable of PHB synthesis, may be genetically modified to possess a portion of the PHB synthesis pathway (e.g., β-ketothiolase and acetoacetyl-CoA reducatase), while excluding PHB synthase functionality. Increased amounts of crotonyl-CoA, which is converted to crotonic acid and then to propylene via crotonyl-CoA thioesterase and 4-OD, respectively, may result in increased propylene yields.

TABLE 1 Exemplary PHB Synthase Sequences Genbank Accession # Gene Name Amino Acid Sequence (SEQ ID NO: #) gi|53719168 poly-beta- MQQLFESWLGAWRSFADPARAAAGDAPSPSPSPFAAFQPPQPFAFAMPAMPPMPDWS hydroxybutyrate GAAASFAGLAPVASVPPARLQKLQADYSRDCLALIQQASAATPTVPELKDRRFSADAWKASP polymerase AHGFAAAWYLLNARYLQELADALETDPKTRERIRFTVQQWTAAASPSNFLALNPEAQKNLV [Burkholderia ETQGESLRLGMMNLLADMQRGKISQTDESQFVVGKNLAVTPGAVVYENDLIQLIQYTPTTA pseudomallei TVFERPLLIVPPCINKFYILDLQPENSLVAHALSCGHQVFLVSWRNADASVAHKTWDDYIDEG K96243] LLAAIDVVQQVSGREQINTLGFCVGGTMLATALAVLAARGEHPAASMTLLTSMLDFSDTGIL DVFVDEAHVQMREQTIGGKGGAPAGLMRGVEFANTFSFLRPNDLVWNYVVDNYLKGRTP APFDLLYWNGDSTSLPGPMYAWYLRNTYLENKLREPDALTVCGEPVDLSRIDVPTFIYGSRED HIVPWQTAYASTSLLTGPLKFVLGASGHIAGVINPPAKRKRSYWSYDASAKELPESANDWLD AAVEHPGSWWPVWIEWLDQYGGKKVKPRAHLGCARFPVIEPAPGRYVLQRD (SEQ ID NO: 54) gi|296445329 poly(R)- MTAGRRSTAGAKRRPPHLRDAAETKLVVEEPPPAENADVSLLEAPRVNGARVAAAAPAQKK hydroxyalkanoic KTGKVRAKPRAPVQEPPVQEPPVREPTAEAPREAMIAIATPPAAPTAEERRLELQAAAALVA acid GAAAELTEAQRRSLMGQLQALEPAPPAPAPVLTPAAPPEIPAAPQRKPARDYQAGAAESTA synthase, HDFEIIAENLARLVDQGRKALAAAVGGMEPGDTRSELASNVADATKTLGAVAERWMAKPE class I QAVAAQADLLTGLSAIWSQTLRRFSGADVPPVVPADPSDKRFSAPEWRDNPFFDCLRQSYA [Methylosinus LTTHWASEAVERTDGLDPQTKSKAAFYTRLIASALSPSNFVATNPELLRATLDARGENLVRG trichosporium MKMLTEDLSAGRGMLKLRQSDESKFELGVDMASTPGKVVYRTPVMELIQYAPSTETVYERP OB3b] LLIVPPWINKFYVLDLNREKSFVRWATGKGLTVFVVSWVNPDESQADKGFDAYMKEGVLAA LDAVQKATGAPHVAAAGYCVGGTMLAATLAYLAEKGDDRIDSVTFLATQVDFTDAGDMQ VFIDEARLAALDEAMSRTGYLEGVKMATTFNMLRPNELFWTYFVNNYMKGVEPAAFDLLT WNSDCTRIPRANHLFYLRYCYIENALSQGRMVIDGVTLNLRKVKIPIYELAAKEDHIAPARSVF TGAKYFGGEVRYVLAGAGHIAGVVNPPDKHKYQYWTGGPPMGAYEDWVKAAKESKGSW WDDWHDWITSQAPEQVAARIPGEGGLKALCDAPGDYVRVRA (SEQ ID NO: 55) gi|296445763 Poly-beta- MVVVDFAGRAGRLQEPAAEASPNPPAPVEEPPRRAYPIGEVPSFDTLDRAARAVAARFTQG hydroxybutyrate VSPLAQMSAWLDFATHLALAPGRQIELGLAAAQSAAALAYFSAETLAGRAEAGPFVSEPHD polymerase RRFVDPGWDAPPFSLWKQGFLATEHWWRLATRETRGMTRSDAARVGFMAEQWLGAFSP domain SNIAWANPAVVERTREEGGANLVRGAENFAEDLLRLVLRAPNGHGEFEVGVDVAATPGEVI protein FRNELIELIQYGPSTETVHAEPILIVPAWIMKYYVLDLRPQNSLVRYLTAQGFTVFMISWRNPG [Methylosinus PEDRDLTFDDYRVSGVLAALSAIDAVRPGSKVHACGYCLGGTLLSIAAAAMARDGDARLASIS trichosporium LLAAQTDFSEPGELMLFVDAAQVAFLEDMMWDQGVLDTRQMAGAFAALRSNDLVWGKA OB3b] VQDYLLGERRKPNDLMAWSADQTRMPYRMHSQYLRGLFLENRLTAGRYAVEGEVVALKDI SAPMFVVGTTSDHIAPWRSVYKASLFTDCELTFVLTSGGHNAGVVSEPGRSGRSYHVGLRRP GDFYVGPDKWLAAARLAEGSWWPEWAAWLAARSGDERVAPPTMGARGYPPLYPAPGRY VHQA (SEQ ID NO: 56) gi|52841292 phbC gene MSAMKGLEKQCCPKKRILTQSQELQEGVDFSCCAPADRGTSDNFFPFFTRLVQANLAKWTA product GISPAAIGSSYSTWLWQLAQSPGVLWELAFYPVFHAKDCINNIVCVERAADGKDVRFKKDS [Legionella WQPMPWRLFAEGFLQMEDWWRRATTDVPGLPNQVERTVSFWARQCLDALSPSNFVWS pneumophila NPDLFHEAMRTGGLNLIQGGQIALEDWLEKLTGAPPTGSEHFIPGKQVAITPGRVVFQNHLI subsp. ELIQYEAQTKTVYKEPILILPAWIMKYYILDLSPHNSLVKWLVSQGHTVFIISWRNPDKEDQDL pneumophila GMDDYYRQGAMAAIDAVSTLFPETKINLMGYCLGGTLAMITAAAMGRDKDERLNSLTLLA str. AQGDFTEAGELMLFVTESQVDFLKSMMREQGYLDTKQMAGSFQMLRAYDLIWSKMVQD Philadelphia YMHGMRRGMIDLTAWNADATRMPYKMHSEYLEKLFLRNDFAEGRYTVEGKPVAAENIKLP 1] VFAVSTEKDHVAPWQSVYKIHLMTEGDVTFVLTGGGHNAGIISEPGHPGRSYRVHEQKQGE AYLNPESWLAMAERREGSWWREWNEWLVQQNTKKRIASSVMNPSLPEAPGTYVLQK (SEQ ID NO: 57) gi|148359804 PHA synthase MKKTTEKPINKTKEMPASSKKEILNPLEQTPSPEQSDPIFRFIDKLYQANLGKLTAGISPAALGT [Legionella AYYSWFAQLLQSPGSMLRLAYYPLLHANDYLSNLFKYDKPRDGKDVRFHTENWSYYPWRL pneumophila WAEQFLQFEDWCLQASSKVPGIPLHVKRTVTFSTRQILDALSPSNFVLTNPDLLQETIRSNGQ str. Corby] NLIRGTELAFQDFVEKITGSPPAGVENFIPGKQVAVTKGKVVYSNHLIELIQYTPQTEKVYKEPI LILPAWIMKYYILDLLPENSLVNWLVRQGHTVFIVSWRNPTKEDRNLGLDDYYKLGAMDAIN AVSNAIPHTKIHLMGYCLGGTLALLTAAAMAHDHDNRLKTLSLLAAQGDFIDAGELLLFITKS EVSFLKSMMWEQGYLDTKQMSGTFQMLRSYDLIWSKMVQDYMHGTQRGMIPLLAWNA DATRMPYKMHSEYLEKLFLNNDFAEGRFILDGKPVVGENIRIPAFVVSTEKDHVAPWKSVYK THLLINSDITFVLTNGGHNAGIVSEPGHEGRYYRIRERKMDSTYLDPTNWLKKAELREGSWW IAWHDWLVNHSSQKQVSAPKLDKKLPNAPGKYVLQK (SEQ ID NO: 58) gi|326404096 poly(3- MPGFATFDRLSRAMFARISQGVSPLAVADAWTDWALHLELALGKQEALAMRAATFLLRLG hydroxyalkanoate) FWLPRAAIGEPENPPLRPPEGDRRFADEGWSAYPFNAIVQGFLVADDWWREATRQVPGLV polymerase RRHEAEVAFMARQILDIAAPTNIPWLNPEIIRRTLQEGGFNLLRGWSNWIEDAERLLAGQGP [Acidiphilium YGAEAFPVGEVVATTAGKVVYRNELMELIQYAPATAKVTAEPVLIVPAWIMKYYILDLTPETS multivorum LVRYLVTNGHTVFIISWINPDRHDRNVGLDDYRRHGVMAALDAVSRIVPDRAIHACGYCLG AIU301] GTILAIAAATMARDHDDRLASLTLLAAQTDFADAGDLMLFLDERQFLLLEDLMWDQGYLDT RQMAGAFQALRSNELVWSRLIRNYMLGERDRMTPLSAWNSDQTRMPARMHSEYLRGLFL ENRLSAGRFAVEGRVIALRDIRVPLFAVATTRDHIAPWRSVYKIALFADTDITFVLASGGHNV GVVNPPNQSIGTFQILTRRHGERYVDPDTWATLAPERQGSWWPAWRDWIGGVGTGCAA DPPPMAAPSRGLPVLGDAPGAYVHER (SEQ ID NO: 59) gi|330826932 poly-beta- MPDTFSAAALADQLDTQFHAALARRFFSLSPAAGMLAASDWALHLAVSPGKCMALARLAL hydroxybutyrate RQSEELAGYARERMTAGADPQLRHGVQPPAQDRRFAAPEWQQWPFNYMHQSFLLTQQ polymerase WWAAATHGVKGVEKHHENVVAFAARQLLDVFSPGNQLATNPVVLQRTLQQGGANLLRG domain- ALNAADDLQRLAAGKPPAGTEDFVVGRDVAVTPGKVVLRNRLVELIQYTPTTEAVHPEPVLI containing VPAWIMKYYILDLSPHNSLIRYLVDQGHTVFCLSWKNPGYEDRDLGLDDYLKLGFHAALDAV protein NAIVPKRKVHATGYCLGGTLLAIAAAAMARDGDARLASLSLFAAQTDFSEPGELGLFIDESQV [Alicycliphilus NLLEAQMTQTGYLKASQMAGAFQMLRSYDLLWSRLVNEYLLGERTPMNDLMAWNADAT denitrificans RMPARMHAQYLRRLLLDDDLSRGRYPVGGKPVSLSDITLPIFMVGTLTDHVAPWRSVHKLH K601] HLTTTEITFALTSGGHNAGIVNPPGNPRRHYQLRTRPAGGNYMAPDDWLAAAPAAQGSW WPAWQEWLQARSGKPVAPPHMGARGYKAGADAPGHYVLEK (SEQ ID NO: 60) gi|222110509 poly-beta- MSNVATHGKADLAQPEACRPDTLDTLANAWRARSTGGLSPAAGLLAWYDWALHLSLSPG hydroxybutyrate KQRSLIEKGLHKQQRLARYALRVASAHDCPTCIEPLEQDRRFAAPAWQQWPFNVIHQGFLL polymerase QQQWWHNATTGVRGVSRHHENMVTFAGRQWLDMWSPSNFIWTNPEVLHAITQSGGA domain- NLWRGAMNFLEDARRLALDDAPAGVEGFEVGKDVAVTPGKVVFRNHLIELIQYSPTTPDVH containing AEPVLIVPSWIMKYYILDLSPHNSMVKYLVDQGHTVFILSWKNPTAADRDLGLEDYRWLGV protein MDALDAVTAIVPERKVQAVGYCLGGTLLAIAAAAMARDGDERLQSLTLLASETDFRESGEIAL [Acidovorax FIDDSQLAWLEAGMWDKGYLDGKQMAGAFQMLNSRDLIWSRRVREYLLGERQTFNDLM ebreus TPSY] AWNADVTRMPYRMHSEYLRRLYLDNDLAEGRYRVGGRPVALADIEVPMFIVGTVRDHVAP WPSVYKMHLLSDAELTFVLTSGGHNAGVVSEPGHPRRSFQIATRAAGDRYIDPQLWRAETP MNEGSWWPAWQQWLAQRSAGRVAPPAMGGTQAPLGDAPGTYVAMR (SEQ ID NO: 61) gi|39937303 phaC gene MMLQTSPPAPSAASQQIQSPARHHGPSDSDRTLHATLAPLTGGLSPTALTLAYADWLSHLF product WAPTQRMDLVNDALRRGTQLAEASIGQTAPWSLIAPQPQDRRFSAPEWREPPFNLMAQG [Rhodopseudomonas FLLAEQWWHDATTNIRGVSEQNEKIVEFATRQMLDVWAPSNFALTNPEVLRRTVSTEGRNL palustris ADGFRNWWEDLLELMAHEPKHDFVVGKDVAVTPGKVVYSNKLIELIQYAPATETVRPEPILI CGA009] VPAWIMKYYILDLSPHNSLVKYLTEQGYTVFMISWRNPTAADRDVSLEDYRRLGVMAALDTI GAILPDRPVHAVGYCLGGTMLSIAAAAMGRDGDSRLKSITLFAAQTDFTEAGELTLFINESQV AFLEDMMWNRGVLDTTQMSGAFQILRSNDLIWSRLVHEYLMGVRSEPNDLMAWNADAT RMPYRMHSEYLRKLFLDNDLAEGRYVVDGRPIALSDIHTPIFVVGTQRDHVAPWRSTFKIHLL ADADVTFCLTGGGHNAGIVSPPSPKAHGYQVMTKEADGPYVGPDDWLKQAPHAEGSWW TEWVHWLGTRSGEPVAPPRIGLPDVDPGALPNAPGSYVLQT (SEQ ID NO: 62) gi|172063316 poly-beta- MKASVTRAPRGNESIRPSGAVEPESSMTQCPQAGTTPAEQWNRAAHANVAAMTFGLSPV hydroxybutyrate SLALAMLDWGAHLAVSPGKCFDLAMQACVAAVAPAADQCEAEAGEADPTGLAVHAGQA polymerase DPRFAAPAWAGWPFHVYRDSFLSIQRWWRDATHGVPGVERHHEELVGFAARQWLDACS domain- PGNFLATNPVVLDATMCSGGANLAAGALNWLEDAKALLERAGGTHAHDARTYLPGRDVAI containing TPGRVVWRNALCELLQYEPTTARVAREPILIVPSWIMKYYILDLQPHNSLIRFLVDAGYTVFAV protein SWRNPGAEARDLGLDDYLRDGCMAALDAARSVCGGAVHTVGYCLGGTLLAIVAAALARDG [Burkholderia RQHEALRSVTLLAAQTDFSEPGELGLFIDASELSALDALMWRQGYLDGAQMSAAFQLLNAR ambifaria DLIWSRMMSEYLLGTRTKPNDLMSWNADTTRMPYRMHSEYLTRLFLDNDLAVGRYCVDG MC40-6] RPVALSDIDVPTFVVGTERDHVSPWGSVYKLHLLTHHALTFVLTSGGHNAGIVSEPGHPGRH YRRATREPGAPYRSRHDFVRGTTAVDGSWWTCWRDWLHERSSGDVPARTPAAGFDAAP GTYVLET (SEQ ID NO: 63) gi|1730539 PHAC_METEX MGTERTNPAAPDFETIARNANQLAEVFRQSAAASLKPFEPAGQGALLPGANLQGASEIDEM RecName: TRTLTRVAETWLKDPEKALQAQTKLGQSFAALWASTLTRMQGAVTEPVVQPPPTDKRFAH Full = Poly(3- ADWSANPVFDLIKQSYLLLGRWAEEMVETAEGIDEHTRHKAEFYLRQLLSAYSPSNFVMTNP hydroxyalkanoate) ELLRQTLEEGGANLMRGMKMLQEDLEAGGGQLRVRQTDLSAFTFGKDVAVTPGEVIFRND polymerase; LMELIQYAPTTETVLKRPLLIVPPWINKFYILDLNPQKSLIGWMVSQGITVFVISWVNPDERH Short = PHA RDKDFESYMREGIETAIDMIGVATGETDVAAAGYCVGGTLLAVTLAYQAATGNRRIKSATFL polymerase; TTQVDFTHAGDLKVFADEGQIKAIEERMAEHGYLEGARMANAFNMLRPNDLIWSYVVNNY AltName: VRGKAPAAFDLLYWNADATRMPAANHSFYLRNCYLNNTLAKGQMVLGNVRLDLKKVKVP Full = PHA VFNLATREDHIAPALSVFEGSAKFGGKVDYVLAGSGHIAGVVAPPGPKAKYGFRTGGPARGR synthase; FEDWVAAATEHPGSWWPYWYKWLEEQAPERVPARIPGTGALPSLAPAPGTYVRMKA AltName: (SEQ ID NO: 64) Full = Polyhydroxy- alkanoic acid synthase gi|16125629 poly-beta- MVETLSANLARAAVTAQGAIAEAALRQADRPAALTPDPFHVAPALNEVMTRLAAQPDRLM hydroxybutyrate RAQADLFGQYMELWQTAARRAAGEDVAPVVAPAAGDKRFNDPDWASNPMFDLMKQSY polymerase LLSSNWLNGLIAEVDGVDPATKRRVEFFTKMLTDAFSPSNFLISNPAALREVVQTQGQSLVR [Caulobacter GMENFAADLERGGGQLAISQTDLAKFKVGENVATAPGKVVYQNDILQLLQFDPTTDTVCEIP crescentus LLIFPPWINKFYIMDLRPENSMIRWLTAQGFTVFVASWVNPDQTLAAKTFEDYMVEGIYDA CB15] AQQVMTQCGVDRVNTVGYCIGGTLLSVALAHMAARGDKRINSATFFAAQQDFAEAGDLLL FTNEEWLQSIEQQMDQAGGFLPSQSMADTFNALRGNDLIWSFFVSNYLMGKEPRPFDLLF WNADQTRMPKALHLFYLRNFYKDNALTTGKLSLGGERLDLSKVKIPIYVQSSKDDHIAPYRSV YRGARAFGGPVTFTMAGSGHIAGVINHPDARKYQHWTNSELPADVSEWIAGAHEHPGSW WPHWAAWLKARSGDQVPARDPAKGKLKPLEDAPGSFVLVKSQP (SEQ ID NO: 65) gi|170744156 unnamed MLATSPTQSSAAIPARPPALRLVPPVAAAGPVRDAAEPGDDLHDAGQAIDQAAHAAVARLT protein GGLSPAALANAWLDWSVHAAFSPGKQAELAAKAFRKGQRLQSFLWRNLLVGAQAEPCIEP product LPQDHRFDDPAWRTWPFCLYQQGFLLTQQWWHNATTGVRGVARAHEEIVAFTARQMLD [Methylobacterium GVSPSNHPLTNPVVLNATLASGGANLVLGALNALEDARRGLAGLKPAGAERFAVGREVAVT sp. 4- EGEVVYRNDLIELIQYAPKTGAVRPEPVLIVPAWIMKYYILDLSPENSLVRHLVGQGFTVFMIS 46] WRNPGPADRDVSFDDYRRLGVMAALDAVSAIRPGRAVHAAGYCLGGTLLSIAAATMARDG DARLASLTLFAAQVDFTEAGELTLFINESQISFLESLMRSEGVLDSKQMAGAFQLLRSNDLIW SRIVNSYLLGRREAVTDLMAWNADATRMPARMHAEYLRRLFLDNDLAEGRLRVEGRPVAL TDIRAPIFAVGTEKDHVAPWRSVFKLTLMTDADVTFLLASGGHNAGIVSEPGHRGRHYRVHS RAATDRYVDPDSWLDLARLEQGSWWPEWASWLAERSGPPEPPPPMGLPGAPTLGPAPG RYVREA (SEQ ID NO: 66) gi|146340526 Poly-beta- MTDVPNNTNPQKTFDAEAFATNIARAMESSGKALAAYLKPRETGEVQDRPPTELTEVVKSFT hydroxybutyrate AVADYWLSDKDRASDIQTKLAKGYLDLWGSAARRLAGEEAPPAISPSPRDKRFADPEWKSN polymerase QFYDFVMQAYLLTTQWAQDLVHNAEGLDPHTRKKAEFYVNQITNALAPSNFVMTNPEVM [Bradyrhizobium RQTVASSGDNLVRGMQMLAEDIEAGKGTLKIRQSDPANLEVGVNMATTPGKVIFQNEMM sp. ORS QLIQYAPTTETVLRTPLLIVPPWINKFYILDLRPEKSFIKWCVDQGLTVFVISWVNPDKKLGTKT 278] WEDYMKEGPLTAMDVIEKVTGEMKVHTMGYCVGGTMLATTLAWLADKRRQRVTSATFLA AQVDFTHAGDLLVFVDETQISALERDMQASGVLEGSKMAMAFNMLRSNDLIWSYVVNNYL KGQPPQAFDLLHWNSDATRMSAANHSYYLRNCYLENRLTTGTMVLDNTLLDLSKVKVPVY NLATREDHIAPADSVLYGSQFFGGPVKYVLSGSGHIAGVVNPPSSGKYQYWTNDQIHDISLK DWMKGAQEHKGSWWPDWREWLGQLDPEQVPARSVGSEAYPPIEDAPGSYVRVRA (SEQ ID NO: 67) gi|126463677 unnamed MSDMKWNAEGAPAYGQALDRAARAAIAGMTRGLAPSVLATAALDWMMHLAAAPGKQA protein ELWEKAATASAALMQAGLQPHEAPVRDRRYASEAWNRQPFAALRDSFLLTEDWWQTATT product GLRGMDRAHEAALSFSVRQMLDVWSPSNNPFLNPEVLARTTETRGANLMQGAMNFAGD [Rhodobacter MARLATGVPMDEGGFRIGETLAATPGKVVLRTHLMELIQYSPTTREVHPEPVLIVPAWIMKY sphaeroides YILDLSEQNSLVRWLVAQGFTVFMISWRNPESEDRDLGLIDYLDQGPRAALKAIQTITGAPKV ATCC 17029] HAAGYCLGGTLLSIMAARMAHDHDERLASMTLFAAQVDFSEAGELALFISEAQVALLEDM MWHQGYLDSDQMSGAFTLLRSNDLIWSRMIHEYMMGERPHPNDLMTWNADSTRMPYR MHSEYLRQLFLENRFAEGKFELEGHALSLTELRLPILAVGTETDHVAPWRSVFKIQRLTETETT FVLTSGGHNAGIVSEPGHPRRHFRIATTGRDDPYRDADEWFAETAPVEGSWWPAWGAWL AERSTPKGKLPPMGNARGGYPALCEAPGTYILQR (SEQ ID NO: 68) gi|359401847 putative MKGLKIMVEAQIPGVGDPLDGLAETMDRAAGAMIAQATFGLSPATLAQAVSDWMLHLAA poly-beta- SPGKQTQLAAKALRKMTRLGDYAMRSATDAQAARAIEPLPQDRRFADPAWASAPFNLVSQ hydroxyalkanoate AFLLNQQWWHAATTGITGVTAHHEEMVAFAVRQWLDTVAPTNFLATNPVLQQRILETGG synthase QCLADGLRNWMTDVEALMRGLPPAGTEAFQVGETLATAEGKVVYRNRLMELIQYAPTTEQ [Novosphingobium ARPEPILIVPAWIMKYYILDLSPENSLVQWLTAQGFTVFMISWHNPGSTDRDLEMADYLQLG pentaromativorans PMAALDAVAAITGGASVHAAGYCLGGTLLAIAAAAMARDGDDRLASLTLLAAQTEFCEPGE US6-1] LGLFIDEGQLSLLENMMWGRGYLDSAQMGGAFQMLRSNDLVWSRVLTTYLMGEREPMN DLMAWNADGTRMPYAMHSQYLRRLFLEDDLAEGRFQVNGRPIALSVLRWPMFVVGTERD HVAPWRSVFKIHRLTGAPIDFVLTSGGHNAGIVSEPGHPGRSYRLLTREADGAALDPDAWLD AAPRHEGSWWTAWGDWLAKLSGNAGTPPPMGATDKGYAPLADAPGHFVLER (SEQ ID NO: 69) gi|581529 PHA synthase MLDHVHKKLKSTLDPIGWGPAVTSVAGRAVRNPQAVTAATAEYAGRLAKIPAAATRVFNA [Rhodococcus NDPDAPMPVDPRDRRFSDTAWQENPAYFSLLQSYLATRAYVEELTEAGSGDPLQDGKARQ ruber] FANLMFDALAPSNFLWNPGVLTRAFETGGASLLRGARYAAHDILNRGGLPLKVDSDAFTVG ENLAATPGKVVFRNDLIELIQYAPQTEQVHAVPILAAPPWINKYYILDLAPGRSLAEWAVQH GRTVFMISYRNPDESMRHITMDDYYVDGIATALDVVEEITGSPKIEVLSICLGGAMAAMAAA RAFAVGDKRVSAFTMLNTLLDYSQVGELGLLTDPATLDLVEFRMRQQGFLSGKEMAGSFD MIRAKDLVFNYWVSRWMKGEKPAAFDILAWNEDSTSMPAEMHSHYLRSLYGRNELAEGLY VLDGQPLNLHDIACDTYVVGAINDHIVPWTSSYQAVNLLGGDVRYVLTNGGHVAGAVNPP GKRVWFKAVGAPDAESGTPLPADPQVWDEAATRYE HSWWEDWTAWSNKRAGELVAPP AMGSTAHPPLEDAPGTYVFS (SEQ ID NO: 70) gi|227823933 poly-beta- MKTGDRPVLAADRARQAGSARAIAAQSALPCENDGADGEAFRAIDRMREALSATATGGLS hydroxybutyrate PAALTLAFFDWSIHLASAPGKRMELAHMAAQNWGLLLTYMAAAATRPDAPPCIEALPGDN polymerase RFRAEGWQKQPYTVWAQAFLLGQQWWHNVTRNVPGMTPHHEDVVSFTARQWLDVFS domain PSNIPFANPEVIHKAMETGGANFTQGFRNWLEDVGRLATKQRPVGTEAFRVGHDTAATPG protein KVVYRNHLIELIQYAPATEEVLAEPILIVPAWIMKYYILDLSPHNSLIRYLVAQGHTVFCISWRN [Sinorhizobium PSAKDRDLTLDDYRRLGILAALDAVSAIVPERKIHATGYCLGGTLLAIAAAAMARTEDQRLAS fredii VTLFAAQTDFSEPGELALFIDHSQLHFLDSLMWHSGCLSADQMAGAFQLLRTNDLVWSRLV NGR234] HDYLIGKRTPMTDLMAWNADPTHMPYRMHAEYLQRLYLDNELAAGRFIVDGRPAHLQNIR VPMFVVGTERDHVAPWHSVYKIHYLTDTDVTFVLTSGGHNAGIVSEPDHPGRGFRIALTRES DSSVSADEWVAAATSKDGSWWPDWVEWLAGHSAPKRVTPPVIGAPERGYPPIDDAPGTY VHQR (SEQ ID NO: 71) gi|332526305 poly-beta- MHNPNVAAAVLADPALDPALAAARRLDSHFHAAVAPAFSGLSPISLALAWQDWALHLATQ hydroxybutyrate PATALALVARAQQSWLQAWGEMLGQAEPGNGDARFAAPAWRQWPWAPVVHGWHAT polymerase- ERWWQDATDLRGVDPHHREVVRFFARQWLDMLAPSNAGLANPEVLQRTAERGGANLVD like protein GTLHALDGWRRQHGLEPLRTPERRYEPGVDVAVTPGQVVWRNHLVELIQYLPLTASVQAEP [Rubrivivax VFIVPSWIMKYYILDLSPHNSFVKWLVEQGHTVFILSWRNPDESDALLAMQDYLELGIFDPLA benzoatilyticus QIARMIPGRRVHACGYCLGGTLLSLAAAALARPGRIARAELLPELASVSLLAAETDFTEPGEM JA2] GVLIDESQVTLLEDMMAERGFLTGAQMAGSFAYLHSRDQVWSRRLREFWLGEPDSPNDL MAWNADLTRMPAAMHSEYLRRCYLRNEIAEGRFPVEGRAVSLSDIAAPMFVVGTEKDHVS PWKSVYKIHRLADTTITFVLTSGGHNAGIVSEPGHANRRYRMATREVDGAWVDPEAWAEQ APRHEGSWWTAWHEWLLAQGSGETAKARTPAKADVVCAAPGTYVYQCWRD (SEQ ID NO: 72) gi|154253888 unnamed MTKNKKGNTSSTIVPALDMQAHMAWAQAWSSISPESSLLAWTDWASHLANSPGKQSELL protein AFAGSLSEQWMSLLKKSLVSPDQEVASPEPSPTNDRRFNDPAWDQWPYNLFRSSFLIQSKW product WEQATQGVWGVDPQHERLLAFGAKQWLEMVSPTNSALFNPVVLKKTIEEQGANLARGM [Parvibaculum SNFLDDLRRQLSGEPPAGTENFVVGRDVAVTEGKVVLRNQLIELIQYTPTTEKVHPEPILIIPA lavamentivorans WIMKYYVLDLSPHNSLIRYLVAQGHTVFCISWKNPDAGDRDLGMDEYLEFGLRAALDAVTSI DS-1] VPEQGIHAAGYCLGGTLLAIGASAMARDGDTRLVSVSLLAAQTDFSEPGELSLFINESQVALL EASMAQTGYLTSDQMSGAFQLLRSYDLIWSRMIDEYLLGDRRPMTDLMAWNADGTRLPA KMHSQYLRRLYLNNDLSAGRYPVTGRPVSVGDIAVPVFCVGTASDHIAPWRSVYKLHLLSSA ELTFVLTTGGHNGGIVSEPGRGKRQYQIHTRAAGDGYMAPDEWQATAQTHLDSWWPAW SAWLRERSGEGVAPPLMGAESRGYPTICDAPGKYVRS (SEQ ID NO: 73) gi|53716799 phbC gene MDTRHAPESGAPDAPLPAHPPASYAPESPYRIFDLAKEASVAKLTSGLSPASLQLALADWLIH product LAAAPGKRAELATLALRHAALLGQYLLEAATGRTPAAPAQPSPGDRRFRAGAWQLEPYRFW [Burkholderia HQSFLLAEQWWRAATRDVPGVSPHHEDVVAFSARQMLDTFAPANYVATNPEIAQRTALT mallei ATCC GGANLAQGVWNYLDDVRRLITKQPPAGAEQFELGRNLATTPGRVVFRNHLIELLQYSPTTPD 23344] VYAQPVLIVPAWIMKYYILDLSAHNSLIRYLVGEGHTVFCISWRNVDASDRDLSLDDYRKLGV MDALDTIGAIVPGEKIHATGYCLGGTLLSIAAAAMANTGDDRLASITLLAAQTDFAEPGELQL FIDDSEIHFLESMMWERGYLGAHQMAGSFQLLMSNDLIWSRVIHDYLLGERTPMIDLMAW NADSTRMPYRMHSEYLRHLFLDNDLATNRYVIDGQTVSVHNIRAPFFVVGTEHDHIAPWRS VYKIHYLSGSDVTFVLTAGGHNAGIVSEPGHAKRHYRMKMTAAAAPSISPDEWLAGATDFE GSWWPAWHAWLARHSSPQRVAPPPLGKPGAHTLGDAPGTYVFQK (SEQ ID NO: 74) gi|365891729 putative MSDAKSAAEDANSVAREFVREDHELDKAFSAVLARLTGGISPAALSMAYLDWACHLAAAPQ poly-beta- RQLDIARDAWQGARQLAERSLHFADSNRVPWDLIKPQANDHRFSKPQWGMQPFNLFAQ hydroxybutyrate AFLLGEDWWHKATTNIRGVDPANEAVVDFSLRQLLDMFAPSNFAATNPEVVEKIFQSGGE polymerase NLVFGWQNWLSDLMQVLQPGQASRSAEFVPGETVATAPGKVVFRNQLIELIQYAPTTAEV (Poly(3- RPEPILIVPAWIMKYYILDLSQHNSLVRYLTDQGFTVFMISWRNPDAKDRDIAFDDYRSMGV hydroxybutyrate) MAALSEIGKIVPGAQIHAAGYCLGGTLLSITAAAMAREGDTRLKTITLFAAQTDFTEAGELTLFI polymerase) NESQVAFLEDMMWERGYLDTTQMAGAFQLLRSNDLIWSRVSRDYLLGERAHPSDLMAW (PHB/PHA NADATRLPYRMHSEYLRKLFLNNDLAEGRYRVEGKPVSLSDIHTPMFVVGTLRDHVAPWKS polymerase) TYKIHYEVDADVTFVLASGGHNAGIVAPPHEQGHSHQVRTKAADAPYLGPDEWQSTSPRIE (PHB/PHA GSWWPTWLDWLAQRSGPLDAPPRLGTEGSHELGNAPGEYVHS (SEQ ID NO: 75) synthase)(Poly(3- hydroxyalkanoate) polymerase) (Polyhydroxy- alkanoic acid synthase) [Bradyrhizobium sp. STM 3809] gi|338984011 Poly(R)- MAEQQNPRTEAPNLPDPAAFSRTMADIAARSQRIVAEWLQRQHEADVAIDPLNIGSAFME hydroxyalkanoic MMARLMANPASLIEAQIGFWQDYVTLWQHSTRRIMGIETQPVVPPDPRDKRFQHEEWKE acid NEIFDFIRQSYLLSARFVQDVVRRVDGLDPKTAQKVDFYARQFVDAMSPSNFALTNPQVLRK synthase, TAETGGENLLRGLSNLLRDLEAGRGQLHIRMTDAEAFSVGGNIAVTPGKVVYRNELMELIQY class I APATTTAHKTPLVIFPPWINKFYILDLRPKNSFIRWAVEQGHTVFVASWVNPDERLAEKDFA [Acidiphilium DYMKLGVFAALDAIEQATGERQVNAIGYCLGGTLLAATLAVMARRRDSRIKSATFLVTLTDF sp. PM] ADVGELGVFIDEEQLAALEDRMNRRGYLEGSAMATTFNMLRSNDLIWSFVVNNYLLGNETF PFDLLYWNSDSTRMPAAMHSFYLRNMYQKNLLSQADAITLDGTPVDLRRIKVPVYFLSCRED HIAPWASTYRATQLMAGPKRFVLAASGHIAGVINPPGSGKYNHFVNATLPANAEDWFAGA TEVAGSWWPDWQRWIAAQGRGEVPARTPGDGALPALADAPGDYVKVRSA (SEQ ID NO: 76)

In certain embodiments, the present disclosure provides a non-naturally occurring C₁ metabolizing bacterium; a non-naturally occurring obligate C₁ metabolizing organism; a non-naturally occurring C₁ metabolizing organism, wherein the non-naturally occurring C₁ metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally CO utilizing bacterium naturally possesses the ability to utilize CO; wherein the non-naturally occurring C₁ metabolizing bacterium or organism converts a C₁ substrate to propylene, and further wherein the non-naturally occurring C₁ metabolizing bacterium or organism has a functional β-ketothiolase and/or a functional acetoacetyl coenzyme A reductase, or has a functional β-ketothiolase and/or a functional acetoacetyl coenzyme A reductase activity (i.e., has enzymes that perform the function of β-ketothiolase and acetoacetyl coenzyme A reductase).

In certain embodiments, a non-naturally occurring C₁ metabolizing organism according to any of the embodiments disclosed herein is a C₁ metabolizing bacterium that has a functional β-ketothiolase activity and a functional acetoacetyl coenzyme A reductase activity.

In certain embodiments a non-naturally occurring C₁ metabolizing organism according to any of the embodiments disclosed herein is a C₁ metabolizing bacterium that does not produce a substantial amount of polyhydroxybutyrate.

β-ketothiolase and acetoacetyl coenzyme A reductase are enzymes in a PHB synthesis pathway that catalyze the condensation of two acetyl-CoA molecules to acetoacetyl-CoA and reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA, respectively (see FIG. 1). β-ketothiolase is an enzyme that initiates biosynthesis of PHB in most bacteria. A C₁ metabolizing organism may naturally possess β-ketothiolase and acetoacetyl coenzyme A reductase as part of an endogenous PHB synthesis pathway. Alternatively, a C₁ metabolizing organism may be genetically modified with exogenous nucleic acids encoding β-ketothiolase and acetoacetyl coenzyme A reductase. These enzymes allow a C₁ metabolizing organism to produce 3-hydroxybutyryl-CoA, a precursor of propylene in an engineered biosynthetic pathway via crotonyl-CoA and crotonic acid as disclosed herein (i.e., 3-hydroxybutyrl-CoA is substrate of crotonase) (see FIG. 1). Exogenous nucleic acids encoding β-ketothiolase and acetoacetyl coenzyme A reductase are expressed in a sufficient amount to produce propylene. In a specific embodiment, β-ketothiolase is encoded by phaA or phbA. In another specific embodiment, acetoacetyl coenzyme A reductase is encoded by phaB, hbd, or phbB. Exemplary β-ketothiolase and acetoacetyl coenzyme A reductase amino acid sequences are provided in Tables 2 and 3, respectively.

TABLE 2 Exemplary β-ketothiolase Amino Acid Sequences Genbank Gene Name/ Accession # Organism Amino Acid Sequence (SEQ ID NO: #) gi|52209583 acetyl-CoA MTDVVIVSAARTAVGKFGGSLAKIAAPELGASVIRAVLERAGVKPEQVSEVILGQVLTAGSG acetyltransferase QNPARQALIAAGLPNAVPGMTINKVCGSGLKAVMLAANAVVAGDAEIVVAGGQENMSA [Burkholderia APHVLPGSRDGFRMGDAKLVDSMIVDGLWDVYNKYHMGITAENVAKEYGITREAQDQF pseudomallei AALSQNKAEAAQKAGRFDDEIVPIEIPQRKGEPLRFATDEFVRHGVTAESLASLKPAFAKEG K96243] TVTAANASGINDGAAAVLVMSAKKAEALGLEPLARIKAYANAGVDPSVMGMGPVPASRR CLERAGWSVGDLDLMEINEAFAAQALAVHKQMGWDTSKVNVNGGAIAIGHPIGASGCRI LVTLLHEMLKRDAKRGLASLCIGGGMGVALALERP (SEQ ID NO: 77) gi|296445575 acetyl-CoA MTTEIVIVSAARTAVGSFNGAFGATPAHELGAVAVKAAIERAGLAPADIDEVILGQVLGAA acetyltransferase QGQNPARQAAIKAGVPQEKTAFGINQVCGSGLRAVALAAQQIQAGDASVIVAGGQESMS [Methylosinus LSQHSAHMRAGTKMGDVKFVDTMIVDGLTDAFNNYHMGITAENVAAKWQISRAEQDAF trichosporium AVASQNKAEAAQKAGKFKDEIVPFTVSTRKGDVIVDQDEYIKHGVTLEGVAKLKPAFTKEGT OB3b] VTAANASGLNDGAAALVVMSAAEAARRGLTPLARIASWATAGVDPSVMGSGPIPASRKA LEKAGWKIGDLDLVEANEAFAAQALAVNKDLGWDPAIVNVNGGAIAIGHPIGASGARVLT TLLYELARRGGKRGLATLCIGGGMGVALTIER (SEQ ID NO: 78) gi|296444966 acetyl-CoA MSAVDPIVIVGAARTPIGALLGELKNATAPQLGAAAIRAATERAGLAPERVDDVLMGCVLS acetyltransferase AGLGQAPARQAALGAGLADTTGCVTINKMCGSGMKALMLAHDQLLAGSSRAIVAGGME [Methylosinus SMSNAPYLLGRARVGYRMGHGRLIDHMFLDGLEDAYDEGKLMGAFAEDCATTHQFTREK trichosporium QDDYATASLRRAQQAAASGAFDWETTPVATHDRKTSATVTRDELPASAKIENIASLKPAFR OB3b] DGGTVTAANSSAISDGAAALALMRRSEAERASLAPLAIVRAHATHAGPPHLFPIAPIGAIAKL CERAGWPLTSVDLFEINEAFAVVVLAAMRELYLPHEKVNVHGGACALGHPIGASGARIVVT LLAALRKYDLRRGVAALCIGGGEATAMAIETIV (SEQ ID NO: 79) gi|296445504 acetyl-CoA MPEAYIYDAVRTPRGRGKPNGSLHEVSSLGLAVAALSALKQRNRLDGAPVDDVILGCVDPV acetyltransferase GEAGGDIARAAAIASGFGYEVPGVQINRFCASGLDAVNFAAAQIMSGQHELTIGGGVESM [Methylosinus SRVGIGASGGAWPADPAIAIPSYFMPQGVSADLIATKYGFSRNDVDAYAMQSQQRAARA trichosporium WEEQRFARSVTPVKDVNGLTILDHDEHMRPSTDMQSLGALKPAFAFLAEQAGFDAVAIQ OB3b] AHPDVEKINYVHHAGNSSGIVDGAAAVLLGSKEAGEKAGLTPRARIRAFANIGSEPALMLT GPVDVTKKLLAKAGMTFGDIDLVEVNEAFAAVVLRFLQAFSLDDSKVNVNGGAIALGHPLG ATGAMLVGTALDELERSGKGVALVTLCIGAGMGTATIIERV (SEQ ID NO: 80) gi|23502628 phbA-1 gene MSDPKSIVIASAARTAVGAFNGAFANVPAHELGAVAIKAALERAGVDAADVDEVILGQVLT product AGEGQNPARQAAMGAGCPKETTAFAINQLCGSGLRAVALGMQQIVSGDAKIIVAGGQES [Brucella suis MSMAPHCAYLRSGVKMGDFKMIDTMLKDGLTDAFHGYHMGITAENIARQWQLSRSEQ 1330] DEFALASQHKAEAAQKAGRFDEEIVPFTVKARKGDVVVSADEYIRPGTTMEVLAKLKPAFD KEGTVTAGNASGINDGAAAVVLMDAGEAARRGVKPLARIVSWATAGVDPSIMGTGPIPA TRKALEKAGWSVGDLDLVEANEAFAAQSCAVVRDLGLNPEIVNVNGGAIAIGHPIGASGAR VLTTLLYEMERRDAKRGLATLCIGGGMGVALCVERD (SEQ ID NO: 81) gi|260682683 acetyl-CoA MGVMNMREVVIASAARTAVGSFGGAFKSVSAVELGVTAAKEAIKRANITPDMIDESLLGG acetyltransferase VLTAGLGQNIARQIALGAGIPVEKPAMTINIVCGSGLRSVSMASQLIALGDADIMLVGGAE [Clostridium NMSMSPYLVPSARYGARMGDAAFVDSMIKDGLSDIFNNYHMGITAENIAEQWNITREEQ difficile DELALASQNKAEKAQAEGKFDEEIVPVVIKGRKGDTVVDKDEYIKPGTTMEKLAKLRPAFKK CD196] DGTVTAGNASGINDGAAMLVVMAKEKAEELGIEPLATIVSYGTAGVDPKIMGYGPVPATK KALEAANMTIEDIDLVEANEAFAAQSVAVIRDLNIDMNKVNVNGGAIAIGHPIGCSGARILT TLLYEMKRRDAKTGLATLCIGGGMGTTLIVKR (SEQ ID NO: 82) gi|147904014 acetyl-CoA MNSGEETVVIISAARTPIGSFNGALSTLPAHTLGSTVIKEVLKRATIKPEEVSEVIFGQVLTAG acetyltransferase AGQNPARQASVAAGVPYSIPAWSCQMICGSGLKAVSLGAQSIKTGEADIVVAGGMENMS 2 QAPHLVHMRAGVKAGDVSLQDSIICDGLNDAFYKYHMGITAENVAKQWQITREEQDQLA [Xenopus VQSQNRTEAAQKAGYFDKEIVPVSVPSRKGPVEVKVDEFPRHGSNVEAMSKLKPYFLKDGS laevis] GTVTPANASGINDGAAAVVLIKESEARRRGLTPMARIVASAQAGLDPSIMGVGPIAAIRKA VEKAGWSLDDIDLFEINEAFAAQALAVVKDLGLNPEKVNCQGGAVALGHPLGMSGCRILV TLLYALERTGGKKGVAALCIGGGMGIAMCVERTS (SEQ ID NO: 83) gi|358447737 acetyl-CoA MRDVVIVAARRTAIGTFGGGLSSLSADQLGTAVIKALMEETGVAGDQINEVVLGQVLTAGV acetyltransferase GQNPARQSAINAGIPASVPAMTINKVCGSGLKAVHMAVQAIRCGDAEMMIAGGQESMS [Marinobacter QAPHVLPNSRNGQRMGNWSMVDTMIKDGLWDAFNDYHMGITAENIVEKYGISRDEQD manganoxydans EFAAASQQKAAAAREAGYFDGQIVPVSIPQRKGDPIVVDRDEGPRDGVTAEGLGKLRAAF Mnl7-9] KKDGTVTAGNASSLNDGAAAVMVCSAEKAKELGLTPIATIKAYANAGVDPTIMGTGPIPAS QRCLKLAGWSTEDLDLVEANEAFAAQAISVNRDMGWDTGKVNVNGGAIALGHPIGASGC RILVSLLHEMVRRDVHKGLATLCIGGGMGVALAVER (SEQ ID NO: 84) gi|294011684 acetyl-CoA C- MPLSDIVITAAKRTAVGGFMGAFGSTPAHELGRTAILAALAQAGVAPEEVDEVILGQVLTA acetyltransferase GQGQNPARQAAVNAGIPVERTAIGVNQLCGSGLRAVALAAQAIRAGDARIMVAGGQES [Sphingobium MSLAPHAQYLRGGAKMGPISLVDTMTHDGLTDAFNNYHMGITAENLAEKYQISREAQDV japonicum FSVGSQNKAEAARASGRFKDEIAPVTVKGRKGDTIVDTDEYIRAGATLEAMQSLRPAFRKD UT26S] GTVTAANASGINDGAAALVLMSAEEAAKRDALVLARIASFATCGVDPSIMGIGPAPASRQA LAKAGWSLADLDLIEANEAFAAQALAVGQELGWDAEKVNVNGGAIAIGHPIGASGARVLT TLIYEMQKRDAKKGLATLCIGGGMGVAMCIER (SEQ ID NO: 85) gi|212696567 hypothetical MTRKVVIASAARTPVGSFGGALKSQSAADLGIVAAKAAIERAGIKPEDIDETVLGCVLQAGL protein GQNVARQISLGAGIPETTPAMTINKVCGSGLRTVSLAAQMILAGDVDVVLAGGAESMSNA ANHYDR0_01105 PFLLNEARWGARMGNKKLVDEMITDGLWDVYNDYHMGVTAENVAEKYGITREMQDDL [Anaerococcus AAVSQQRASKARAEGRFKDEIAPVEIKDRKGNVTVVEDDEYIRDGVTQEGISKLRPAFIKDG hydrogenalis TVTAANASGINDGAACLVVMSEEKAKELGVKPLATIVSYASAGVDPKVMGTGPIPSSKKAL DSM 7454] EKAGWKVEDLDLVESNEAFAAQSYAVRNEMGFDPEKTNVNGGAIAIGHPIGGSGARILTTL LFEMQKRDSKKGLATLCIGGGMGTALVVER (SEQ ID NO: 86) gi|351728760 acetyl-CoA MEDIVIVSAARTAVGKFGGALAKTPATELGAIVIREAIARAGLSSDQIGEVIMGQVLAAGVG acetyltransferase QNPARQASMKAGVAKETPALTINAVCGSGLKAVMLAAQAVAWGDSEIVVAGGQESMSL [Acidovorax APHVLPGSRDGQRMGDWKLIDTMIVDGLWDVYNQYHMGITAENVAKAHGITREMQDA radicis N35] LALGSQQKAAAAQDAGKFVDEIVGVSLAQKKGDPILFNADEYLNRKTNAEALAGLRPAFDK AGSVTAGNASGLNDGAAAVVVMSAKKAAALGLKPLARIAAFGTSGLDPATMGMGPVPAS RKALQRAGWNAADVDLFELNEAFAAQACAVNKELAIDPAKVNVNGGAIAIGHPIGASGCR ILVTLLHEMQRRDAKKGLAALCIGGGMGVSLALER (SEQ ID NO: 87) gi|295687835 acetyl-CoA MSDVVIVSAARTPVGSFNGALSSLPASELGRVAIEAAISRAGLQPSDVDEVILGQVLQAGAG acetyltransferase QGPARQASVKAGIPVESPAWSLNQLCGSGLRAVALAAQQIAAGDAAVVVAGGQESMSQ [Caulobacter APHAQNLRGGQKMGDLSFVDTMIKDGLWDAFHGYHMGQTAENIASRWQITRADQDAF segnis ATCC AVASQNKAEAAQKAGKFDAEIAPVTIKGRKGDTVVDKDEYIRHGVTLESISGLKPAFTKEGS 21756] VTAANASGLNDGAAALVLMSAEEAQKRGLKPLARIASWANAGVEPEIMGTGPIPASKKAL EKAGWTVADLDLVESNEAFAAQSLCVVRELGLDPAKVNVNGGAIAIGHPIGASGARVLTTL LHEMKRSGAQKGLATLCIGGGMGVAMCVEAV (SEQ ID NO: 88) gi|374996374 unnamed MRDVVIVSAVRTPVGSFCGALGQIPAAELGAIAVKEAINRAGITPEQVDEVILGNVLQAGLG protein QNPARQASIKAGIPQEVPSWTLNKVCGSGLKTVVCAAQAIMTGDADIVVAGGMENMSLA product PYVLTKARTGYRMGNDTVIDSMINDGLTDAFNNYHMGITAENIAEQFNISREEQDRYSVRS [Desulfosporosinus QNRAEAAIIAGKFNEEIVPVSIPQRKGDPVVVSQDEFPRFGATYEAIAKLRPAFKKDGTVTAA orientis NASGINDGAAAIVVMAKEKAEELGLTPLATIKSWASAGVDPKIMGTGPIPASRKALEKAGLS DSM 765] IDDIDVVEANEAFASQTLSVAQGLNLDPEKTNVNGGAIALGHPVGASGTRILVTLLHEMKRS NAHRGLATLCIGGGQGVALIVER (SEQ ID NO: 89) gi|330941564 acetyl-CoA MHDVVIVAATRTAVGSFQGSLASVAAVDLGAAVIRQLLARTGVDGAQVDEVIMGQVLTA acetyltransferase GAGQNPARQAAIKAGLPFSVPAMTLNKVCGSGLKALHLATQAIRCGDAEIIIAGGQENMSL [Pseudomonas SNYVLPGARTGLRMGHASMVDTMITDGLWDAFNDYHMGITAENLAQQYDISREAQDEF syringae pv. AALSQQKALAAIEAGRFVDEITPILIPQRKGDPLSFATDEQPRAGTTAETLAKLKPAFKKDGT pisi str. 1704B] VTAGNASSLNDGAAAVMLMSAARAEQLGLPVLARIAAYANAGVDPAIMGIGPVSATRRCL NKAGWSLADLDLIEANEAFAAQSLSVGKELGLDPQKLNVNGGAIALGHPIGASGCRVLVTL LHEMIRRDVKKGLATLCIGGGQGVALALER (SEQ ID NO: 90) gi|27375337 atoB gene MPMSDDVVIVSAARTPVGSFNGAFATLPAHDLGAVAIKAALERGGIEPGRVSEVIMGQILT product AAQGQNPARQASIAAGIPVESPAWGVNQLCGSGLRTVALGYQALLNGDSEIVVAGGQES [Bradyrhizobium MSMAPHAQYLRGGVKMGALEFIDTMIKDGLWDAFNGYHMGNTAENVARQWQITRAQ japonicum QDEFAVASQQKAEAAQKAGKFNDEIVPVTIKTRKGDVVVSADEYPRHGATLDAMAKLRPA USDA 110] FEKDGTVTAGSASGINDGAAAVVLMTAKQAAKEGKKPLARIVSWAQAGVDPKIMGSGPIP ASRAALKKAGWNVGDLDLIEANEAFAAQACAVNKDLGWDTSKVNVNGGAIAIGHPVGAS GARVLVTLLHEMQKRDSKKGLATLCIGGGMGIAMCLARD (SEQ ID NO: 91) gi|163738904 Acetyl-CoA C- MTNVVIASAARTAVGSFGGAFAKTPAHDLGAAVLQAVVERAGIDKSEVSETILGQVLTAAQ acetyltransferase GQNPARQAHINAGLPQESAAWSLNQVCGSGLRAVALAAQHIQLGDAAIVCAGGQENMT [Phaeobacter LSPHAANLRAGHKMGDMSYIDTMIRDGLWDAFNGYHMGQTAENVAEKWQISREMQD gallaeciensis EFAVASQNKAEAAQKAGKFADEIAAFTVKTRKGDIIVDQDEYIRHGATIEAMQKLRPAFAK BS107] DGSVTAANASGLNDGAAATLLMSADDAEKRGIEPLARIASYATAGLDPSIMGVGPIYASRK ALEKAGWSVDDLDLVEANEAFAAQACAVNKDMGWDPAIVNVNGGAIAIGHPIGASGCR VLNTLLFEMKRRDAKKGLATLCIGGGMGVAMCVERP (SEQ ID NO: 92) gi|7766963 A Chain A, SIVIASAARTAVGSFNGAFANTPAHELGATVISAVLERAGVAAGEVNEVILGQVLPAGEGQ Unliganded NPARQAAMKAGVPQEATAWGMNQLCGSGLRAVALGMQQIATGDASIIVAGGMESMS Biosynthetic MAPHCAHLRGGVKMGDFKMIDTMIKDGLTDAFYGYHMGTTAENVAKQWQLSRDEQDA Thiolase From FAVASQNKAEAAQKDGRFKDEIVPFIVKGRKGDITVDADEYIRHGATLDSMAKLRPAFDKE Zoogloea GTVTAGNASGLNDGAAAALLMSEAEASRRGIQPLGRIVSWATVGVDPKVMGTGPIPASRK Ramigera ALERAGWKIGDLDLVEANEAFAAQACAVNKDLGWDPSIVNVNGGAIAIGHPIGASGARIL NTLLFEMKRRGARKGLATLCIGGGMGVAMCIESL (SEQ ID NO: 93) gi|218892041 atoB gene MQDVVIVAATRTAVGSFQGSLAGIPAPELGAAVIRRLLEQTGLDAGQVDEVILGQVLTAGS product GQNPARQAAIKAGLPVGVPAMTLNKVCGSGLKALHLGAQAIRCGDAEVIVAGGQENMSL [Pseudomonas APYVMPGARTGLRMGHAKLVDSMIEDGLWDAFNDYHMGITAENLAEKYGLSREEQDAF aeruginosa AAASQQKAIAAIEGGRFRDEITPIQVPQRKGEPLSFDTDEQPRAGTTVEALAKLKPAFRKDG LESB58] SVTAGNASSLNDGAAAVLLMSAAKAKALGLPVLARIASYASAGVDPAIMGIGPVSATRRAL DKAGWSLEQLDLIEANEAFAAQSLAVGRELGWDAARVNVNGGAIALGHPIGASGCRVLVT LLHEMIRRDAKKGLATLCIGGGQGVALTLARD (SEQ ID NO: 94) gi|374292777 phbA3 gene MTEVVIAGAARTPIGSFNGALSAVPAHVLGEVAIREALARAKTDAAEVDEVILGQILTAGQG product QNPARQAAVNAGIPVEATAMGINQLCGSGLRAVALGYQAIKNGDADVLVVGGQESMSM [Azospirillum APHVMHLRNGTKMGSAELLDTMLRDGLTDAFHGYHMGTTAENVAQKWQLTREEQDAF lipoferum 413] AAASQQKAEAAQKAGRFKDEIVPVTIKGRKGDVVVSDDEYPKHGTTPESLAKLRPAFSKDG TVTAGNASGINDGAAALVLMTAENAAKRGVTPLARIVSWATAGVDPAIMGTGPIPASRKA LEKAGWTVDDLDLIEANEAFAAQALSVNKDLGWDTSKVNVNGGAIALGHPVGASGARVL TTLLYEMQKRDAKKGLATLCIGGGMGIALCVQRD (SEQ ID NO: 95) gi|17546351 phbA gene MTDVVIVSAVRTAVGKFGGSLAKIPAPELGAAVIREALSRAKVAPDQVSEVIMGQVLTAGS product GQNPARQALIKAGLPDMVPGMTINKVCGSGLKAVMLAANAIVAGDADIVVAGGQENMS [Ralstonia AAPHVLPGSRDGFRMGDTKLIDSMIVDGLWDVYNQYHMGITAENVAKQYGITREAQDAF solanacearum AVASQNKAEAAQKSGRFNDEIVPILIPQRKGDPIAFAQDEFVRHGATLESMTGLKPAFDKA GM11000] GTVTAANASGLNDGGAAVVVMSAARAKELGLTPLATIRAYANAGVDPKVMGMGPVPAS KRCLSRAGWSVGDLDLMEINEAFAAQALAVHQQMGWDTAKVNVNGGAIAIGHPIGASG CRILVTLLHEMQKRDAKKGLASLCIGGGMGVALAVERP (SEQ ID NO: 96) gi|221198056 Acetyl-CoA MTDVVIVSAARTAVGKFGGSLAKVAAPELGATVIRAVLERAGVKPEQVSEVIMGQVLTAGS acetyltransferase GQNPARQSLIKAGLPSAVPGMTINKVCGSGLKAVMLAANAIVAGDAEIVVAGGQENMSA [Burkholderia APHVLPGSRDGFRMGDAKLVDTMIVDGLWDVYNQYHMGITAENVAKEYGITREEQDAF multivorans AALSQNKAEAAQKAGRFNDEIVPVSIPQRKGEPLQFATDEFVRHGVTAESLAGLKPAFAKD CGD2M] GTVTAANASGINDGAAAVLVMSAQKAQALGLTPLARIKAYANAGVDPSVMGMGPVPAS RRCLERAGWTPGDLDLMEINEAFAAQALAVHKQMGWDTSKVNVNGGAIAIGHPIGASG CRILVTLLHEMVKRDAKRGLASLCIGGGMGVALAVERP (SEQ ID NO: 97) gi|163852882 acetyl-CoA MAASEDIVIVGAARTPVGSFAGAFGSVPAHELGATAIKAALERAGVSPDDVDEVIFGQVLT acetyltransferase AAAGQNPARQAAIAAGIPEKATAWGLNQVCGSGLRTVAVGMQQIANGDAKVIVAGGQE [Methylobacterium SMSLSPHAQYLRGGQKMGDLKLVDTMIKDGLWDAFNGYHMGQTAENVAQAFQLTREQ extorquens QDQFAVRSQNKAEAARKEGRFKEEIVPVTVKGRKGDTVVDTDEYIRDGATVEAMAKLKPA PA1] FAKDGTVTAANASGLNDGAAALVLMSASEAERRGITPLARIVSWATAGVDPKVMGTGPIP ASRKALEKAGWKPADLDLIEANEAFAAQALAVNKDMGWDDEKVNVNGGAIAIGHPIGAS GARVLITLLHELKRRDAKKGLATLCIGGGMGVAMCVERV (SEQ ID NO: 98) gi|169344179 acetyl-CoA MREVVIASAVRTALGSFGGSLKDVPAVDLGALVIKEALNKAGVKPECVDEVLMGNVIQAGL acetyltransferase GQNPARQAAVKAGLPVEIPSMTINKVCGSGLRCVALAAQMIKAGDADVIVAGGMENMS [Clostridium QGPYVLRTARFGQRMGDGKMVDAMVNDALTDAFNGYHMGITAENIAEQWGLTREMQ perfringens C DEFAANSQIKAEAAIKAGKFKDEIVPVVIPQRKGDPIVFDTDEFPRFGTTAEKLAKLRPAFKK str. JG51495] DGTVTAGNASGINDGAAALVVMSAEKAKELGVTPICKIVSYGSKGLDPSIMGYGPFYATKK ALEGTGLKVEDLDLIEANEAFAAQSLAVAKDLEFDMSKVNVNGGAIALGHPVGASGARILV TLLHEMMKRDAKRGLATLCIGGGMGTALIVER (SEQ ID NO: 99) gi|345869447 acetyl-CoA MSENIVIVDAGRTAIGTFGGSLSSLPATELGTTVLKALLARTGIAPDQIDEVILGQVLTAGVG acetyltransferase QNPARQTTLKAGLPHAVPAMTINKVCGSGLKAVHLAMQAVACGDADIVIAGGQECMSQ [Thiorhodococcus SSHVLPRSRDGQRMGDWKMVDTMIVDGLWDAFNQYHMGVTAENIAKQFGFTREAQD drewsii TFAAESQQKAEAAIKAGRFKDEIVPVSIPQRKGDPLVVDTDEFPRAGTTAAGLGKLRPAFDK AZ1] EGTVTAGNASGINDGAAMVVVMKESKAKELGLKPMARIVAFASAGVDPAIMGTGPIPAST KCLEKAGWTPADLDLIEANEAFAAQAMSVNKEMGWDLSKVNVNGGAISLGHPIGASGAR VLVTLLHEMQHRDAKKGLATLCIGGGQGVALAVERL (SEQ ID NO: 100)

TABLE 3 Exemplary Acetoacetyl Coenzyme A Reductase Amino Acid Sequences Genbank Gene Name/ Accession # Organism Amino Acid Sequence (SEQ ID NO: #) gi|52209584 acetoacetyl- MSQRIAYVTGGMGGIGTSICQRLHKDGFRVVAGCGPNSPRRVKWLEDQKALGFDFYAS CoA EGNVGDWDSTKQAFDKVKAEVGEIDVLVNNAGITRDVVFRKMTREDWQAVIDTNLTSL reductase FNVTKQVIDGMVERGWGRIINISSVNGQKGQFGQTNYSTAKAGIHGFTMSLAQEVATK [Burkholderia GVTVNTVSPGYIGTDMVKAIRPDVLEKIVATIPVRRLGSPDEIGSIVAWLASEESGFSTGAD pseudomallei FSLNGGLHMG (SEQ ID NO: 101) K96243] gi|296445576 acetoacetyl- MGRTAVVTGGTRGIGEAISKALKAAGYNVAATYAGNDEAANKFKDATGIPVYKFDVSDY CoA DACAAALAAIETDLGPVDVLVNNAGITKDRLFHKMELAQWRAVIDTNLNSLFNVTRPVI reductase NGMRDRGFGRIIVISSINGQKGQAGQTNYSASKAGDIGFVKALAQESAAKGITVNAIAPG [Methylosinus YIATEMVKAVPQEVLDKHIIPHIAVGRLGEPEEIARAVVFLASDEAGFITGSTLTINGGQYLT trichosporium (SEQ ID NO: 102) OB3b] gi|206559226 putative MTKRIAVVTGGMGGLGEAVSIRLNDAGHRVVVTYSPNNTGADRWLTEMHAAGREFHA Acetoacetyl- YPVDVADHDSCQQCIEKIVRDVGPVDILVNNAGITRDMTLRKLDKVNWDAVIRTNLDSV CoA FNMTKPVCDGMVERGWGRIVNISSVNGSKGSVGQTNYAAAKAGMHGFTKSLALEIAR reductase KGVTVNTVSPGYLATKMVTAIPQDILDTKILPQIPAGRLGKPEEVAALVAYLCSEEAGFVT [Burkholderia GSNIAINGGQHMH (SEQ ID NO: 103) cenocepacia J2315] gi|340047249 acetoacetyl- MRKIALITGSKGGIGSAISTQLVSEGYRVIATYYTGNYQCALDWFNEKQFTEDQVRLLELD CoA VTNTEECAERLAKLLEEEGTIDVVVNNAGITRDSVFKKMPHQAWKEVIDTNLNSVFNVT reductase QPLFAAMCEKGFGRIINISSVNGLKGQFGQTNYSAAKAGMIGFSKALAAEGARYGVTVN family protein VIAPGYTLTPMVEQMRAEVLQSIVDQVPMKRLAKPEEIANAVSYLASDAAYITGETLSVN [Vibrio GGLYMR (SEQ ID NO: 104) cholerae HE48] gi|356882300 acetoacetyl- MARVAVVTGGTRGIGEAISVALKNAGYVVAANYAGNDEKAKEFSARTGIAVYKFDVSDF coA reductase DAVKDGIAKISAELGPVDVVVNNAGITRDGVIHRMTPQQWNDVIATNLTSCFNLCRNVI [Azospirillum DGMRERGFGRIVNIGSVNGQAGQYGQVNYAAAKSGIHGFTKALAQEGAAKGVTVNAI brasilense APGYIDTDMVRAVPPNVLEKIVARIPVGRLGKAEEIARGVLFLVGDDAGFITGSTLSINGG Sp245] QHMY (SEQ ID NO: 105) gi|356881146 acetoacetyl- MSQKIALVTGAMGGLGTAICQALAKDGYIVAANCLPNFEPAAAWLGQQEALGFKFYVA coA reductase EGDVSDFESCKAMVAKIEADLGPVDILVNNAGITRDKFFAKMEKAQWDAVIATNLSSLF [Azospirillum NVTQQVSAKMAERGWGRIINISSVNGVKGQAGQTNYSAAKAGVIGFTKALAAELATKG brasilense VTVNAIAPGYIGTDMVMAIREDIRQAITDSVPMKRLGRPDEIGGAVSYLASEIAGYVTGST Sp245] LNINGGLNYQ (SEQ ID NO: 106) gi|119897313 phbB1 gene MSRVALVTGGMGGLGEAICIKLAALGYRVVTTYSPGNSKAAEWLQAMNNMGYGFRGY product PCDVSDFDSCKACIAQVTEEVGPIDVLVNNAGITRDMTFKKMTKADWDAVISTNLDSVF [Azoarcus sp. NMTKQVMDGMVERKWGRVINVSSVNGQKGAFGQTNYSAAKAGMHGFTKALALEVA BH72] RSGVTVNTISPGYIGTKMVMAIPQEILESKILPQIPVSRLGKPEEIAGLVAYLSSDEAAFVTG ANISINGGQHMF (SEQ ID NO: 107) gi|194289469 acetyacetyl- MTQRIAYVTGGMGGIGTAICQRLARDGFRVVAGCGPNSPRREKWLEQQKALGFDFVAS CoA EGNVADWDSTKAAFDKVKAEVGEVDVLINNAGITRDVVFRKMTRADWDAVIDTNLTSL reductase FNVTKQVIDGMADRGWGRIVNISSVNGQKGQFGQTNYSTAKAGLHGFTMALAQEVAT [Cupriavidus KGVTVNTVSPGYIATDMVKAIRQDVLDKIVGTIPVKRLGEPEEIASICAWLASEESGFSTGA taiwanensis DFSLNGGLHMG (SEQ ID NO: 108) LMG 19424] gi|307609363 hypothetical MDKMIAIVTGGTGGIGSAISQRLADSYQVVACYYKDGRHEEAKKWQDEQKQLGYDIDIV protein YGDIAQYSDCEKITSLVMERYGRIDVLVNNAGITKDCSLRKMTPQQWQQVLDANLTSVF LPW_06391 NMTRNVVPVMLERGYGRIISISSINGRKGQFGQCNYASTKAALFGFTKSLALEVASKGITV [Legionella NTVSPGYIETPMLAALKEDVLNSIISSIPVGRLGYPKEIADAVAFLASPDSGFITGANLDVN pneumophila GGQYM (SEQ ID NO: 109) 130b] gi|352104657 acetoacetyl- MTNQAPVAWVTGGTGGIGTAICRSLADAGYLVVAGYHNPDKAKTWLETQRADGYNNI CoA ELSGVDLSDHNACLEGAREIHDKYGPISVLVNCAGITRDGTMKKMSYEQWYEVLDTNLN reductase SVFNTCRSVIEMMLENGYGRIINISSINGRKGQFGQVNYAAAKAGMHGLTMSLAQETAT [Halomonas KGITVNTVSPGYIATDMIMNIPEKVREAIRETIPVKRYGTPEEIGRLVTFLADKESGFITGAN sp. HAL1] IDINGGQFMG (SEQ ID NO: 110) gi|289671313 acetoacetyl- MTSRVALVTGGTGGIGTAICKRLADQGHRVASNFRNEEKARDWQQRMQAQGYAFALF CoA RGDVASSEHARALVEEVEASLGPIEVLVNNAGITRDTTFHRMTAEQWHEVINTNLNSVF reductase NVTRPVIEGMRKRGWGRVIQISSINGLKGQYGQANYAAAKAGMHGFTISLARENAAFG [Xanthomonas VTVNTVSPGYVATDMVMAVPEEVRAKIVADIPTGRLGRPEEIAYAVAFLVAEEAAWITGS campestris NLDINGGHHMGW (SEQ ID NO: 111) pv. musacearum NCPPB 4381] gi|330824321 acetoacetyl- MNTTQRTALVTGGNRGLGAAIARALHDAGHRVIVTHTPGNTTIGQWQQAQATQGYKF CoA AAYGVDVSNYESTQELARRIHADGHRIDILVNNAGITRDATLRKLDKAGWDAVLRTNLDS reductase MFNVTKPFIDPMVERGWGRIVNISSINGSKGQFGQTNYSAAKAGVHGFTKALAQEVAR [Alicycliphilus KGVTVNTVSPGYLATEMVMAVREDMRQKIIDAIPVGRLGQPDEIAALVAFIASEAAAFM denitrificans TGSNVAMNGGQHMY (SEQ ID NO: 112) K601] gi|146278501 acetoacetyl- MSKVALVTGGSRGIGAAISLALKNAGYTVAANYAGNDEAAQKFTAETGIKTYKWSVADY CoA DACAEGIARVEAELGPVAVLVNNAGITRDSMFHKMTREQWKEVIDTNLSGLFNMTHPV reductase WSGMRDRKFGRIINISSINGQKGQAGQANYSAAKAGDLGFTKALAQEGARAGITVNAIC [Rhodobacter PGYIGTEMVRAIDEKVLNERIIPQIPVGRLGEPEEIARCVVFLASDDAGFITGSTITANGGQ sphaeroides YFT (SEQ ID NO: 113) ATCC 17025] gi|67458545 phbB gene MSEIAIVTGGTRGIGKATALELKNKGLTVVANFFSNYDAAKEMEEKYGIKTKCWNVADFE product ECRQAVKEIEEEFKKPVSILVNNAGITKDKMLHRMSHQDWNDVINVNLNSCFNMSSSV [Rickettsia MEQMRNQDYGRIVNISSINAQAGQVGQTNYSAAKAGIIGFTKALARETASKNITVNCIAP felis GYIATEMVGAVPEDVLAKIINSIPKKRLGQPEEIARAVAFLVDENAGFITGETISINGGHN URRWXCal2] MI (SEQ ID NO: 114) gi|94497737 Acetoacetyl- MSRVAIVTGGTRGIGEAISLALKEMGYAVAANYAGNDEKAKAFTDKTGIAAFKWDVGD CoA HQACLDGCAQVAEVLGPVDIVVNNAGITRDGVLAKMSFDDWNEVMRINLGGCFNMA reductase KACFGGMRERGWGRIVNIGSINGQAGQYGQVNYAAAKSGIHGFTKALAQEGAKYGVT [Sphingomonas VNAIAPGYIDTDMVAAVPAPVLEKIVAKIPVGRLGQAHEIARGVAFFCSEDGGFVTGSTLS sp. SKA58] INGGQHMY (SEQ ID NO: 115) gi|28901060 acetoacetyl- MKKVALITGSKGGIGSAISSQLVNDGYRVIATYFTGNYECALEWFNSKGFTKDQVRLFELD CoA VTNTAECAEKLAQLLEEEGTIDVVVNNAGITRDGVFKKMTAQAWNDVINTNLNSLFNVT reductase QPLFAAMCEKGGGRVINISSVNGLKGQFGQANYSAAKAGMIGFSKALAYEGARSGVTV [Vibrio NVIAPGYTGTPMVEQMKPEVLESITNQIPMKRLATPEEIAASVSFLVSDAGAYITGETLSV parahaemolyticus NGGLYMH (SEQ ID NO: 116) RIMD 2210633] gi|161522918 acetoacetyl- MSAKRVAFVTGGMGGLGAAISRRLHDVGMTVAVSHTEGNDHVATWLTHEREAGRTF CoA HAFEVDVADYDSCRQCASRVLAEFGRVDVLVNNAGITHDATFVKMTKSMWDAVLRTN reductase LDGMFNMTKPFVPGMIEGGFGRIVNIGSVNGSRGAYGQTNYAAAKAGIHGFTKALALEL [Burkholderia ARHGVTVNTVAPGYLATAMLETVPKEVLDTKILPQIPVGRLGNPDEIAALVAFLCSDAAAF multivorans ATGAEFDVNGGMHMK (SEQ ID NO: 117) ATCC 17616] gi|161524658 acetyacetyl- MSQRIAYVTGGMGGIGTSICQRLSKDGFKVVAGCGPNSPRRVKWLEEQKALGFDFIASE CoA GNVGDWDSTKAAFDKVKAEVGEIDVLVNNAGITRDVVFRKMTHEDWTAVIDTNLTSLF reductase NVTKQVIDGMVERGWGRIINISSVNGQKGQFGQTNYSTAKAGIHGFTMALAQEVATKG [Burkholderia VTVNTVSPGYIGTDMVKAIRPDVLEKIVATIPVRRLGTPEEIGSIVAWLASNDSGFATGAD multivorans FSLNGGLHMG (SEQ ID NO: 118) ATCC 17616] gi|351728759 3-ketoacyl- MSQKVAYVTGGMGGIGTAICQRLHKEGFKVIAGCGPTRDHAKWLAEQKALGYTFYASV (acyl-carrier- GNVGDWDSTVEAFGKTKAEHGTIDVLVNNAGITRDRMFLKMSREDWDAVIETNLNSM protein) FNVTKQVVADMVEKGWGRIVNISSVNGEKGQAGQTNYSAAKAGMHGFSMALAQELA reductase TKGVTVNTVSPGYIGTDMVKAIRPDVLEKIVATVPVKRLGEPSEIASIIAWLASEEGGYATG [Acidovorax ADFSVNGGLHMG (SEQ ID NO: 119) radicis N35] gi|240140211 phaB gene MAQERVALVTGGTRGIGAAISKRLKDKGYKVAANYGGNDEAANAFKAETGIPVFKFDVG product DLASCEAGIKAIEAELGPIDILVNNAGITRDGAFHKMTFEKWQAVIRTNLDSMFTCTRPLI [Methylobacterium EGMRSRNFGRIIIISSINGQKGQAGQTNYSAAKAGVIGFAKALAQESASKGVTVNVVAPG extorquens YIATEMVMAVPEDIRNKIISTIPTGRLGEADEIAHAVEYLASDEAGFVNGSTLTINGGQHF AM1] V (SEQ ID NO: 120) gi|148258780 3-oxoacyl- MARVALVTGGTRGIGAAISKALKAAGHKVAANYGGNDAAAEKFKSETEIPVYKWDVSSF ACP DACAEGIKKVEAELGPVDILVNNAGITRDTAFHKMTLEQWSAVINTNLGSLFNMTRPVIE reductase GMRARKFGRIINISSINGQKGQFGQVNYSAAKAGDIGFTKALALETAKAGITVNVICPGYI [Bradyrhizobium NTEMVQAVPKDVLEKAILPLIPVGRLGEPEEIARAVVFLAADEAGAITGSTLSINGGQYMA sp. BTAi1] (SEQ ID NO: 121) gi|126728325 Acetoacetyl- MARVALVTGGSRGIGEAISKALKAEGYTVAATYAGNDEKAAAFTADTGIKTYKWNVADY CoA ESSKAGIAQVEADLGPIDVVVANAGITRDAPFHKMTPAQWNEVIDTNLTGVFNTVHPV reductase WPGMRERKFGRIIVISSINGQKGQFAQVNYAATKAGDLGIVKSLAQEGARAGITANAICP [Sagittula GYIATEMVMAVPEKVRESIIGQIPAGRLGEPEEIARCVVFLASDDAGFINGSTISANGAQF stellata E-37] FV (SEQ ID NO: 122) gi|15895965 hbd gene MKKVCVIGAGTMGSGIAQAFAAKGFEVVLRDIKDEFVDRGLDFINKNLSKLVKKGKIEEA product TKVEILTRISGTVDLNMAADCDLVIEAAVERMDIKKQIFADLDNICKPETILASNTSSLSITE VASATKRPDKVIGMHFFNPAPVMKLVEVIRGIATSQETFDAVKETSIAIGKDPVEVAEAPG FVVNRILIPMINEAVGILAEGIASVEDIDKAMKLGANHPMGPLELGDFIGLDICLAIMDVLY SETGDSKYRPHTLLKKYVRAGWLGRKSGKGFYDYSK (SEQ ID NO: 123)

In certain embodiments, a non-naturally occurring C₁ metabolizing organism according to any of the embodiments disclosed herein is a C₁ metabolizing bacterium selected from Methylosinus trichosporium strain OB3b, Methylococcus capsulatus Bath strain, Methylomonas methanica 16A strain, Methylosinus trichosporium (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris, Methylacidiphilum infernorum, Methylomicrobium alcaliphilum, or Methylibium petroleiphilum.

In certain embodiments, a non-naturally occurring C₁ metabolizing organism may be a syngas or CO utilizing bacterium that naturally possesses the ability to utilize syngas or CO, such as Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium Woodii, Clostridium neopropanologen Ralstonia eutropha, or Eurobacterium limosum.

In certain embodiments, a non-naturally occurring C₁ metabolizing organism may be a methylotrophic bacterium, such as Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, or Methylobacterium nodulans.

In another embodiment, the present disclosure provides non-naturally occurring microbial organisms that have been genetically modified with a novel metabolic pathway for producing propylene from a carbon substrate. More specifically, the non-naturally occurring microbial organisms include an exogenous nucleic acid encoding 4-oxalocrotonate decarboxylase and convert a carbon substrate to propylene. As described previously, 4-OD is used in a novel propylene biosynthetic pathway to catalyze decarboxylation of crotonic acid to propylene and CO₂ (see FIG. 1). Sources of 4-OD encoding nucleic acid molecules may include any species, prokaryotic or eukaryotic, where the encoded gene product is capable of catalyzing decarboxylation of crotonic acid to propylene and CO₂. Exemplary amino acid sequences of 4-OD are shown in FIG. 2.

In certain embodiments, the present disclosure provides a non-naturally occurring microbial organism containing an exogenous nucleic acid encoding a 4-oxalocrotonate decarboxylase, wherein the non-naturally occurring microbial organism is capable of converting a carbon substrate into propylene.

In certain embodiments, non-naturally occurring microbial organisms that include an exogenous nucleic acid encoding a 4-oxalocrotonate decarboxylase and convert a carbon substrate into propylene may further include an exogenous nucleic acid encoding crotonase, or further include an exogenous nucleic acid encoding a crotonase and an exogenous nucleic acid encoding a crotonyl thioesterase. Depending on the host microbial organism selected, a microbial organism may or may not have endogenous enzyme(s) that would participate with 4-OD in forming a biosynthetic propylene synthesis pathway. As described in detail previously, crotonase catalyzes the dehydration of 3-hydyroxybutyryl-CoA to crotonyl-CoA. Crotonyl-CoA thioesterase catalyzes the conversion of crotonyl-CoA to crotonic acid. For example, if a host microbial organism selected is deficient in crotonase, then exogenous expression of crotonase can be included in the microbial organism. In another example, if a host microbial organism is deficient in crotonase and a thioesterase capable of converting crotonyl-CoA to crotonic acid, then exogenous expression of crotonase and crotonyl-CoA thioesterase can be included in the microbial organism. However, it is understood that exogenous expression of all of these enzymes of a propylene biosynthetic pathway (i.e., 4-OD, crotonase, and crotonyl-CoA thioesterase) may be included, even if the host microbial organism contains at least one of the propylene pathway enzymes (e.g., crotonase or crotonyl thioesterase). Expression of exogenous nucleic acids encoding enzymes of the propylene biosynthetic pathway disclosed herein is in a sufficient amount to produce propylene. Sources of crotonase and crotonyl-CoA thioesterase encoding nucleic acids may include any species, prokaryotic or eukaryotic, where the encoded gene products are capable of catalyzing dehydration of 3-hydyroxybutyryl-CoA to crotonyl-CoA and conversion of crotonyl-CoA to crotonic acid, respectively. Exemplary amino acid sequences for crotonase and crotonyl-CoA thioesterase are shown in FIG. 4 and FIG. 3, respectively.

In a further embodiment, non-naturally occurring microbial organisms as described herein do not have a functional PHB synthase or a substantial amount of functional PHB synthase. As described in detail previously, by inhibiting or reducing PHB function in a microbial organism, propylene synthesis yields may be increased by funneling more 3-hydroxybutyryl-CoA into the propylene pathway via conversion to crotonyl-CoA and to crotonic acid and to propylene (see FIG. 1).

Additionally, if non-naturally occurring microbial organisms as described herein do not possess an endogenous PHB synthesis pathway, then non-naturally occurring microbial organisms may be genetically modified to include an exogenous nucleic acid encoding β-ketothiolase and an exogenous nucleic acid encoding acetoacetyl-CoA reductase to provide the capability of producing 3-hydroxybutyrl-CoA, substrate for the crotonase enzyme in the propylene synthesis pathway described herein. Non-naturally occurring microbial organisms that do possess an endogenous PHB synthesis pathway may also be genetically modified with an exogenous nucleic acid encoding β-ketothiolase and an exogenous nucleic acid encoding acetoacetyl-CoA reductase to increase expression of these enzymes. In a specific embodiment, β-ketothiolase is encoded by phaA or phbA. In another specific embodiment, acetoacetyl coenzyme A reductase is encoded by phaB or phbB. Exemplary β-ketothiolase and acetoacetyl coenzyme A reductase amino acid sequences are provided in Tables 2 and 3, respectively.

Nucleic acid sequences encoding for and amino acid sequences for proteins, protein domains and fragments thereof, for proteins described herein, such as 4-OD, crotonase, crotonyl thioesterase, acetoacetyl coenzyme A reductase, or β-ketothiolase, and domains thereof, that are described herein include natural and recombinantly engineered variants. These variants retain the function and biological activity (including enzymatic activities if applicable) associated with the parent (or wildtype) protein. These variants may have improved function and biological activity (e.g., higher enzymatic activity, improved specificity for substrate) than the parent (or wildtype protein). For example, a variant 4-OD enzyme may be engineered with reduced or eliminated decarboxylation activity on 4-oxalocrotonate, but retains or has increased decarboxylation activity on crotonic acid substrate. Conservative substitutions of amino acids are well known and may occur naturally in the polypeptide (e.g., naturally occurring genetic variants) or may be introduced when the polypeptide is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a polypeptide using well-known and routinely practiced mutagenesis methods (see, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY 2001). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. Deletion or truncation variants of proteins may also be constructed by using convenient restriction endonuclease sites adjacent to the desired deletion. Alternatively, random mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to prepare polypeptide variants (see, e.g., Sambrook et al., supra).

Differences between a wild type (or parent) nucleic acid or polypeptide and the variant thereof, may be determined by methods routinely practiced in the art to determine identity, which are designed to give the greatest match between the sequences tested. Methods to determine sequence identity can be applied from publicly available computer programs. Computer program methods to determine identity between two sequences include, for example, BLASTP, BLASTN (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST family of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.

Assays for determining whether a polypeptide variant folds into a conformation comparable to the non-variant polypeptide or fragment include, for example, the ability of the protein to react with mono- or polyclonal antibodies that are specific for native or unfolded epitopes, the retention of ligand-binding functions, the retention of enzymatic activity (if applicable), and the sensitivity or resistance of the mutant protein to digestion with proteases (see Sambrook et al., supra). Polypeptides, variants and fragments thereof, can be prepared without altering a biological activity of the resulting protein molecule (i.e., without altering one or more functional activities in a statistically significant or biologically significant manner). For example, such substitutions are generally made by interchanging an amino acid with another amino acid that is included within the same group, such as the group of polar residues, charged residues, hydrophobic residues, and/or small residues, and the like. The effect of any amino acid substitution may be determined empirically merely by testing the resulting modified protein for the ability to function in a biological assay, or to bind to a cognate ligand or target molecule.

It is understood that the non-naturally occurring microbial organisms or C₁ metabolizing organisms that have been genetically modified as described herein may lead to the biosynthetic production of propylene, including other pathway intermediates (e.g., crotonate or crotonyl-CoA) and downstream products. Like other alkenes, propylene undergoes addition reactions relatively easily at room temperature due to the relative weakness of its double bond. Through polymerization, oxidation, halogenations and hydrohalogenation, alkylation, hydration, oligomerization, and hydroformylation reactions, which are well known to a person of skill in the art, propylene may be converted into other downstream products (e.g., polypropylene, propylene oxide). These addition reactions may occur spontaneously in the non-naturally occurring microbial organisms or C₁ metabolizing organisms, or the organisms may be further genetically modified to add or enhance addition reaction capability (e.g., to increase conversion to polypropylene or propylene oxide). For example, in methanotrophic bacteria that are genetically modified with a biosynthetic propylene pathway, propylene that is produced may spontaneously be oxidized into propylene oxide via a methane-monooxygenase-catalyzed reaction (see, e.g., U.S. Patent Publication 2002/0168733, U.S. Patent Publication 2003/0203456). Alternatively, the non-naturally occurring microbial organisms or C₁ metabolizing organisms may comprise further genetic modifications to inhibit or reduce endogenous enzyme activity that catalyze an addition reaction (e.g., to inhibit conversion to propylene oxide). In non-naturally occurring organisms that spontaneously convert or are genetically modified to convert propylene into a downstream product (e.g. propylene oxide), there may be little propylene product to recover and measure, and the downstream product (e.g., propylene oxide) may be recovered and measured as a surrogate for propylene production.

Methods of Producing Crotonic Acid or Propylene in Non-Naturally Occurring C₁ Metabolizing Organisms

In certain embodiments, the present disclosure provides methods of producing propylene by culturing non-naturally occurring C₁ metabolizing organisms according to any of the embodiments as described herein (e.g., capable of converting a C₁ substrate into propylene), under conditions sufficient to produce propylene. In a specific embodiment, the non-naturally occurring C₁ metabolizing organisms as disclosed herein produce from about 0.1 grams of propylene/L/day to about 50 grams of propylene/L/day. In another embodiment, the non-naturally occurring C₁ metabolizing organisms as disclosed herein produce about 0.1 g, 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, or 60 g propylene/L/day.

Additionally, the present disclosure provides methods of producing propylene in non-naturally occurring microbial organisms according to any of the embodiments as described herein (e.g., having a partially heterologous propylene biosynthetic pathway), by culturing the non-naturally occurring microbial organisms under conditions sufficient to produce propylene. In a specific embodiment, the non-naturally occurring microbial organisms as disclosed herein produce from about 0.1 grams of propylene/L/day to about 50 grams of propylene/L/day. In another embodiment, the non-naturally occurring microbial organisms as disclosed herein produce about 0.1 g, 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, or 60 g propylene/L/day.

Also disclosed herein are methods of producing crotonic acid by culturing non-naturally occurring C₁ metabolizing organisms under conditions sufficient to produce crotonic acid, wherein the organisms include an exogenous nucleic acid encoding crotonyl-CoA thioesterase. In a specific embodiment, the non-naturally occurring C₁ metabolizing organisms as disclosed herein produces from about 0.1 grams of crotonic acid/L/day to about 50 grams of crotonic acid/L/day. In another embodiment, the non-naturally occurring C₁ metabolizing organism as disclosed herein produces about 0.1 g, 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, or 60 g crotonic acid/L/day.

Codon Optimization

Expression of recombinant proteins is often difficult outside their original host. For example, variation in codon usage bias has been observed across different species of bacteria (Sharp et al., 2005, Nucl. Acids. Res. 33:1141-1153). Over-expression of recombinant proteins even within their native host may also be difficult. In certain embodiments of the invention, nucleic acids (e.g., a nucleic acid encoding 4-OD, crotonyl-CoA thioesterase, or crotonase) that are to be introduced into organisms of the invention may undergo codon optimization to enhance protein expression. Codon optimization refers to alteration of codons in genes or coding regions of nucleic acids for transformation of an organism to reflect the typical codon usage of the host organism without altering the polypeptide for which the DNA encodes. Codon optimization methods for optimum gene expression in heterologous organisms have been previously described (see., e.g., Welch et al., 2009, PLoS One 4:e7002; Gustafsson et al., 2004, Trends Biotechnol. 22:346-353; Wu et al., 2007, Nucl. Acids Res. 35:D76-79; Villalobos et al., 2006, BMC Bioinformatics 7:285; U.S. Patent Publication 2011/0111413; U.S. Patent Publication 2008/0292918).

Transformation Methods

Non-naturally occurring microbial organisms as described herein may be transformed to comprise at least one exogenous nucleic acid to provide the host organism with a new or enhanced activity (e.g., enzymatic activity) or may be genetically modified to remove or substantially reduce an endogenous gene function (e.g., enzymatic activity) using a variety of methods known in the art. Recombinant methods for exogenous expression of nucleic acids in microbial organisms are well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

A non-naturally occurring C₁ metabolizing bacterium; non-naturally occurring obligate C₁ metabolizing organism; non-naturally occurring C₁ metabolizing organism, wherein the organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non-naturally occurring CO utilizing bacterium as described herein may be transformed to comprise at least one exogenous nucleic acid to provide the host with a new or enhanced activity (e.g., enzymatic activity) or may be genetically modified to remove or substantially reduce an endogenous gene function (e.g., enzymatic activity) using a variety of methods known in the art. While genetic engineering tools of C₁ metabolizing organisms are not as extensive as for other microbial organisms (e.g., E. coli), significant advances have been made allowing genetic manipulation of C₁ metabolizing organisms, as summarized below.

Transformation refers to the transfer of a nucleic acid (e.g., exogenous nucleic acid) into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid are referred to as “non-naturally occurring” or “recombinant” or “transformed” or “transgenic” organisms.

Expression systems and expression vectors useful for the expression of heterologous nucleic acids in C₁ metabolizing organisms are known. Vectors or cassettes useful for the transformation of suitable host organisms are available.

Electroporation of C₁ metabolizing bacteria has been previously described in Toyama et al., 1998, FEMS Microbiol. Lett. 166:1-7 (Methylobacterium extorquens); Kim and Wood, 1997, Appl. Microbiol. Biotechnol. 48:105-108 (Methylophilus methylotrophus AS1); Yoshida et al., 2001, Biotechnol. Lett. 23:787-791 (Methylobacillus sp. strain 12S), and US2008/0026005 (Methylobacterium extorquens).

Bacterial conjugation, which refers to a particular type of transformation involving direct contact of donor and recipient cells, is more frequently used for the transfer of nucleic acids into C₁ metabolizing bacteria. Bacterial conjugation involves mixing “donor” and “recipient” cells together in close contact with each other. Conjugation occurs by formation of cytoplasmic connections between donor and recipient bacteria, with unidirectional transfer of newly synthesized donor nucleic acids into the recipient cells. A recipient in a conjugation reaction is any cell that can accept nucleic acids through horizontal transfer from a donor bacterium. A donor in a conjugation reaction is a bacterium that contains a conjugative plasmid, conjugative transposon, or mobilized plasmid. The physical transfer of the donor plasmid can occur through a self-transmissible plasmid or with the assistance of a “helper” plasmid. Conjugations involving C₁ metabolizing bacteria have been previously described in Stolyar et al., 1995, Mikrobiologiya 64:686-691; Motoyama et al., 1994, Appl. Micro. Biotech. 42:67-72; Lloyd et al., 1999, Archives of Microbiology 171:364-370; and Odom et al., PCT Publication WO 02/18617; Ali et al., 2006, Microbiol. 152:2931-2942.

Expression of heterologous nucleic acid molecules in C₁ metabolizing bacteria is known in the art (see, e.g., U .S. Pat. No. 6,818,424, U.S. Patent Publication 2003/0003528). Mu transposon based transformation of methylotrophic bacteria has been described (see, e.g., Akhverdyan et al., 2011, Appl. Microbiol. Biotechnol. 91:857-871). A mini-Tn7 transposon system for single and multicopy expression of heterologous genes without insertional inactivation of host genes in Methylobacterium has been described (see, e.g. U.S. Patent Publication 2008/0026005).

Various methods for inactivating, knocking-out, or deleting endogenous gene function in C₁ metabolizing organisms may be used. Allelic exchange using suicide vectors to construct deletion/insertional mutants in slow growing C₁ metabolizing bacteria have also been described in Toyama and Lidstrom, 1998, Microbiol. 144:183-191; Stolyar et al., 1999, Microbiol. 145:1235-1244; Ali et al., 2006, Microbiology 152:2931-2942; Van Dien et al., 2003, Microbiol. 149:601-609.

Suitable homologous or heterologous promoters for high expression of exogenous nucleic acids may be utilized. For example, U.S. Pat. No. 7,098,005 describes the use of promoters that are highly expressed in the presence of methane or methanol for heterologous gene expression in C₁ metabolizing bacteria. Additional promoters that may be used include deoxy-xylulose phosphate synthase methanol dehydrogenase operon promoter (Springer et al., 1998, FEMS Microbiol. Lett. 160:119-124); the promoter for PHA synthesis (Foellner et al. 1993, Appl. Microbiol. Biotechnol. 40:284-291); or promoters identified from native plasmid in methylotrophs (EP296484). Non-native promoters include the lac operon Plac promoter (Toyama et al., 1997, Microbiology 143:595-602) or a hybrid promoter such as Ptrc (Brosius et al., 1984, Gene 27:161-172). Regulation of expression of an exogenous nucleic acid molecule in the host C₁ metabolizing organism may also be utilized. For example, an inducible/regulatable system of recombinant protein expression in methylotrophic and methanotrophic bacteria has been described in US Patent Publication 2010/0221813.

Methods of screening are disclosed in Brock, supra. Selection methods for identifying allelic exchange mutants are known in the art (see, e.g., U .S. Patent Publication No. 2006/0057726, Stolyar et al., 1999, Microbiol. 145:1235-1244; and Ali et al., 2006, Microbiology 152:2931-2942.

Culture Methods

A variety of culture methodologies may be used for the C₁ metabolizing organisms described herein. For example, C₁ metabolizing organisms, particularly methanotrophic or methylotrophic bacteria, may be grown by batch culture and continuous culture methodologies. In certain embodiments, the cultures are grown in a controlled culture unit, such as a fermentor, bioreactor, hollow fiber membrane bioreactor, or the like.

A classical batch culturing method is a closed system where the composition of the media is set at the beginning of the culture and not subject to external alterations during the culture process. Thus, at the beginning of the culturing process, the media is inoculated with the desired organism or organism and growth or metabolic activity is permitted to occur without adding anything to the system. Typically, however, a “batch” culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures, cells moderate through a static lag phase to a high growth logarithmic phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase are often responsible for the bulk production of end product or intermediate in some systems. Stationary or post-exponential phase production can be obtained in other systems.

The Fed-Batch system is a variation on the standard batch system. Fed-Batch culture processes comprise a typical batch system with the modification that the substrate is added in increments as the culture progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measureable factors, such as pH, dissolved oxygen, and the partial pressure of waste gases such as CO2. Batch and Fed-Batch culturing methods are common and known in the art (see, e.g., Thomas D. Brock, Biotechnology: A Textbook of Industrial Microbiology, 2^(nd) Ed. (1989) Sinauer Associates, Inc., Sunderland, Mass.; Deshpande, 1992, Appl. Biochem. Biotechnol. 36:227, herein incorporated by reference).

Continuous cultures are “open” systems where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in logarithmic phase growth. Alternatively, continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added and valuable products, by-products, and waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials.

Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limited nutrient, such as the carbon source or nitrogen level, at a fixed rate and allow all other parameters to modulate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing the rate of product formation, are well known in the art, and a variety of methods are detailed by Brock, supra.

Culture media must contain carbon substrates for the C₁ metabolizing organisms. Suitable substrates include, but are not limited to C₁ substrates such as methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines (methylamine, dimethylamine, trimethylamine, etc.), methylated thiols, or methyl halogens (bromomethane, chloromethane, iodomethane, dichloromethane, etc.). In certain embodiments, a non-naturally occurring C₁ metabolizing organism of any of the disclosed embodiments is capable of growth on methane, methanol, formaldehyde, formic acid, carbon monoxide, carbon dioxide, methylated amines, methylated thiols, or methyl halogens as a carbon source.

A culture media may comprise a single C₁ substrate as the sole carbon source for the C₁ metabolizing organism, or comprise a mixture of two or more C₁ substrates (mixed C₁ substrates) as multiple carbon sources for the C₁ metabolizing organism. Additionally, some C₁ metabolizing organisms are known to utilize non-C₁ substrates, such as glucosamine and a variety of amino acids for metabolic activity. For example, some Candida species can metabolize alanine or oleic acid (Sulter et al., 1990, Arch. Microbiol. 153:485-489). Methylobacterium extorquens AM1 is capable of growth on a limited number of C2, C3, and C4 substrates (Van Dien et al., 2003, Microbiol. 149:601-609). Alternatively, a C₁ metabolizing organism may be engineered with the ability to utilize alternative carbon substrates. Hence, it is contemplated that a culture media may comprise a mixture of carbon substrates, with single and multi-carbon compounds (mixed carbon sources), depending on the C₁ metabolizing organism selected. In certain embodiments, a C₁ substrate provided in a mixed carbon source may be a primary carbon source for a C₁ metabolizing organism. A carbon source may be added to culture media initially, provided to culture media intermittently, or supplied continuously.

Propylene Separation and Recovery

Propylene or propylene oxide produced by the non-naturally occurring organisms described herein may be dissolved in the liquid phase or present as gas in the headspace of the culture container. Propylene may be mixed with other gases in the headspace, such as O₂, CO₂, H₂O vapor, or methane. Methods for recovering propylene from a gas mixture have been previously described, and include for example, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration (see., e.g., Bai et al., 2000, J. Memb. Sci. 174:67-79; Shi et al., 2006, J. Membr. Sci. 282:115-123; Membranes: Separation of Organic Vapors from Gas Streams, by Ohlrogge and Sturken, Ullmann's Encyclopedia of Industrial Chemistry, 2002, Wiley-VCH Verlag GmbH & Co., KGaA; U.S. Pat. No. 4,348,476; Hou et al., 1984, Applied Microbio. and Biotechnol. 19:1-4; each of the preceding references are incorporated herein by reference, in their entirety). A person skilled in the art can adapt propylene recovery methods used in fluidic cracking process (see, e.g., U.S. Pat. No. 3,893,905; U.S. Pat. No. 6,308,532; U.S. Pat. No. 6,730,142; U.S. Pat. No. 7,875,758) to recover propylene from a fermentation off-gas mixture.

Measuring Propylene Production

Methods for measuring propylene and propylene oxide production are well known in the art and include HPLC (high performance liquid chromatography), GC-MS (gas chromatography-mass spectrometry), GC-FID (gas chromatography-flame ionization detector) and LC-MS (liquid chromatography-mass spectrometry). Methods of measuring propylene and propylene oxide concentration have also been described in, for example, Brown et al., 1963, Anal. Chem. 35:2172-2176; Lin et al., 2000, J. Am. Chem. Soc. 122:11275-11285; Lee and Hwang, J. Membrane Sci. 73:37-45; U.S. Patent Publication 2010/0197986; U.S. Patent Publication 2003/0203456; U.S. Patent Publication 2002/0168733; Stanley and Dalton, 1992, Biocatalysis & Biotransformation 6:163-175; each of which is incorporated herein by reference in its entirety).

Measuring PHB Production

In certain embodiments, the non-naturally occurring organisms as described herein do not produce a substantial amount of polyhydroxybutyrate (PHB). As used herein, “not producing a substantial amount of polyhydroxybutyrate” means that an organism produces at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% less polyhydroxybutyrate as compared to a wildtype organism that has a polyhydroxybutyrate synthesis pathway. Methods for determining PHB concentration are well known in the art. Braunegg et al., (1978, European J. Appl. Microbiol. Biotechnol. 6:29-37) describe a gas chromatographic method for determining PHB concentration comprising a mild acid or alkaline methanolysis of PHB directly without previous extraction from the cells, which is followed by gas chromatography of the 3-hydroxybutyric acid methylester. A quantitative staining method for detecting PHB in viable cells has also been described (see., e.g., Tyo et al., 2006, Appl. Environ. Microbiol. 72:3412-3417). Stopped-flow attenuated total reflection FT-IR spectrometry has been used to determine intracellular PHB content in bacteria (see., e.g., Jarute et al., 2004, Anal. Chem. 76:6353-6358). Additional methods for measuring PHB have been described in Huang and Reusch, 1996, J. Biol. Chem. 271:22196-22202; Henneke et al., 2005, Bioprocess & Biosystems Engineering 27:359-364; Pieja et al., 2011, Appl. Environ. Microbiol. 77:6012-6019; Taguchi et al., 2001, FEMS Microbiol. Letters 198:65-71.

Additional methods for measuring PHB synthesis may include measuring PHB synthase expression (see., e.g., Langenbach et al., 1997, FEMS Microbiol. Lett. 150:303-309; Solaiman et al., 2008, J. Ind. Microbiol. & Biotechnol. 35:111-120) or enzyme activity (see., e.g., Schubert et al., 1988, J. Bacteriol. 170:5837-5847; Liebergesell et al., 1994, Eur. J. Biochem. 226:71-80; Valentin and Steinbuchel, 1994, Appl. Microbiol. Biotechnol. 40:699-709).

EXAMPLES Example 1 Methylosinus Trichosporium Methanotroph Preparation of NMS Media.

-   -   MgSO₄.7H₂O . . . 1.0 g     -   CaCl₂.6H₂O . . . 0.20 g     -   Chelated Iron Solution (see below) . . . 2.0 ml     -   KNO₃. . . 1.0 g     -   Trace Element Solution (see below) . . . 0.5 ml     -   KH₂PO₄ . . . 0.272 g     -   Na₂HPO₄.12H₂O . . . 0.717 g     -   Purified Agar (e.g., Oxoid L28) . . . 12.5 g     -   Distilled deionized water . . . 1.0 L     -   Adjust pH to 6.8. Autoclave at 121° C. for 15 minutes.

Chelated Iron Solution:

-   -   Ferric (III) ammonium citrate* . . . 0.1 g     -   EDTA, sodium salt . . . 0.2 g     -   HCl (concentrated) . . . 0.3 ml     -   Distilled deionized water . . . 100.0 ml     -   *0.5 g of Ferric (III) chloride may be substituted.     -   Use 2.0 ml of this chelated iron solution per liter of final         medium.

Trace Element Solution:

-   -   EDTA . . . 500.0 mg     -   FeSO₄.7H₂O . . . 200.0 mg     -   ZnSO₄.7H₂O . . . 10.0 mg     -   MnCl₂.4H₂O . . . 3.0 mg     -   H₃BO₃ . . . 30.0 mg     -   CoCl₂.6H₂O . . . 20.0 mg     -   CaCl₂.2H₂O . . . 1.0 mg     -   NiCl₂.6H₂O . . . 2.0 mg     -   Na₂MoO₄.2H₂O . . . 3.0 mg     -   Distilled water . . . 1.0 L     -   Autoclave at 121° C. for 15 minutes.

Growth and Conjugations. The procedure for conjugating plasmids from E. coli into methanotrophs was based on the method developed by Martin, H. & Murrell, J. C. (1995), FEMS Microbiol. Lett. 127: 243-248.

Briefly, a mobilizing plasmid to be conjugated was first transformed into E. coli S17-1 using standard electroporation methods. Transformation was confirmed by selection of kanamycin-resistant colonies on LB-agar containing 20 μg/mL kanamycin. Transformed colonies were inoculated into LB media containing 20 μg/mL kanamycin and shaken overnight at 37° C. A 10 mL aliquot of the overnight culture was then collected on a sterile 47 mm nitrocellulose filter (0.2 mm pore size). The E. coli donor strain was washed on the filter with 50 mL sterile NMS media to remove residual media and antibiotic.

In parallel, a sample of the M. trichosporium OB3b recipient strain was inoculated into 100 mL serum bottles containing 20-50 mL NMS media. The headspace of the bottles was then flushed with a 1:1 mixture of oxygen and methane, and the bottles were sealed with butyl butyl rubber septa and crimped. The bottles were shaken continuously in a 30° C. incubator until reaching an OD600 of approximately 0.3. The cells were then collected on the same filter as the E. coli donor strain. The filter was again washed with 50 mL of sterile NMS media. The filter was placed (cells up) on an NMS agar plate containing 0.02% (w/v) proteose peptone and incubated for 24 h at 30° C. in the presence of methane and oxygen. After 24 h, cells were re-suspended in 10 mL sterile (NMS) medium before being concentrated by centrifugation. The pellet was re-suspended in 1 mL sterile NMS media. Aliquots (100 μl) were spread onto NMS agar plates containing 10 μg/mL kanamycin.

The plates were incubated in sealed chambers containing a 1:1 mixture of methane and oxygen maintained at 30° C. The gas mixture was replenished every 2 days until colonies formed, typically after 7-14 days. Colonies were streaked onto NMS plates containing kanamycin to confirm kanamycin resistance as well as to further isolate transformed methanotroph cells from residual E. coli donor cells.

Deletion of phaC. A synthetic cDNA construct of the M. trichosporium OB3b phaC gene was synthesized, incorporating several stop mutations and frame shifts in the 5′ region of the gene. This cDNA construct was cloned into an appropriate vector for conjugation, but lacking an origin of replication that functions in methanotrophs, and introduced into M. trichosporium OB3b using the methods described above. This technique ensures that any kanamycin resistant M. trichosporium OB3b colonies must have been incorporated into the genome by recombination.

Identification of homologous recombination events is well-established in the art, and typically performed by PCR and sequencing using unique primers in the genome and the vector construct to confirm proper insertion. Homologous recombinants are then grown in the absence of selective pressure (e.g., kanamycin) for several generations, and sensitive clones which have lost the resistance marker are identified by replica plating (or equivalent technique). Approximately 50% of sensitive revertants possess the mutated form of the target gene in place of the wild-type version.

Loss of phaC function is confirmed by growing the cells under nitrogen limited conditions and measuring PHB content as described in Pieja A J, Rostkowski K H, Criddle C S, Distribution and selection of poly-3-hydroxybutyrate production capacity in methanotrophic proteobacteria. Microb. Ecol. 2011 October; 62(3):564-73. Briefly, the putative knockout clones inoculated into 100 mL serum bottles containing 20-50 mL NMS media. The headspace of the bottles was then flushed with a 1:1 mixture of oxygen and methane, and the bottles were sealed with butyl rubber septa and crimped. The bottles were shaken continuously in a 30° C. incubator until reaching an OD600 of approximately 0.3-0.6 to ensure the cells are in logarithmic phase growth. Cells are collected by centrifugation at 4,816×g (4,700 rpm) for 8 min, washed once with nitrogen-free NMS media medium, and re-suspended in nitrogen-free NMS medium to induce PHB production. 20-50 mL aliquots of washed cells are then transferred to serum bottles, sealed, and methane and oxygen added as described above. Cultures are then incubated at 30° C. on orbital shakers at 150 rpm. Assays for optical density and PHB production are performed every 1 to 2 h for the first 20 h.

PHB concentration determination. PHB concentration is measured directly via gas chromatography. For each sample, 5 to 10 mg of freeze-dried biomass is weighed out on an analytical balance, transferred to a 12-ml glass vial, and sealed with a polytetrafluoroethylene (PTFE)-lined plastic cap. 2 mL of methanol acidified with sulfuric acid (3%, vol/vol) and containing 1.0 g/L benzoic acid and 2 ml of chloroform are added to each vial. The vials are shaken gently and then heated at 100° C. for 3.5 h. Once the vials cool to room temperature, 1 ml deionized water is added to each. The vials are subjected to vortex mixing for 30 s and allowed to stand until phase separation is complete. The organic phase is analyzed using an Agilent 6890N gas chromatograph equipped with an HP-5 column [containing (5% phenyl)-methylpolysiloxane; Agilent Technologies] and a flame ionization detector (FID). dl-β-Hydroxybutyric acid sodium salt is used as a standard.

Introduction of Propylene pathway. Selected crotonase (SEQ ID NO:32), crotonyl-CoA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO:10) sequences were codon optimized (see Table 4) and synthesized with appropriate promoters. The genes are then cloned and transformed into the phaC knockout strain as described above. Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

TABLE 4 Codon Optimized Sequences for M. trichosporium OB3b Reference Sequence Codon optimized sequence (SEQ ID NO: #) crotonase ATGGAGCTGAATAACGTCATCCTGGAAAAAGAAGGCAAGGTCGCGGTCGTCACGATCAACC GCCCCAAGGCTCTGAACGCGCTCAATAGCGACACCCTCAAAGAGATGGACTACGTCATCGGC GAGATCGAGAACGACTCCGAGGTGCTGGCCGTCATCCTCACCGGAGCGGGCGAGAAGTCGT TCGTGGCTGGGGCGGATATCAGCGAGATGAAAGAGATGAATACCATTGAAGGCCGCAAGTT CGGCATCCTGGGCAACAAGGTGTTCCGTCGCCTGGAGCTGCTGGAGAAGCCGGTCATTGCG GCGGTGAATGGCTTCGCGCTCGGCGGTGGCTGCGAGATCGCGATGTCGTGCGACATCCGCA TCGCCTCGTCGAACGCCCGCTTCGGACAGCCGGAAGTCGGCCTCGGCATCACGCCCGGATTC GGCGGCACTCAGCGCCTCAGCCGCCTGGTGGGCATGGGCATGGCTAAGCAGCTGATCTTCA CGGCGCAGAACATCAAGGCTGACGAGGCGCTCCGCATCGGCCTGGTCAACAAGGTGGTGG AGCCGTCGGAACTCATGAACACGGCGAAAGAGATTGCGAACAAAATCGTGTCGAATGCGCC GGTGGCGGTCAAGCTGAGCAAGCAGGCGATCAACCGTGGCATGCAGTGCGATATCGACACT GCGCTCGCCTTCGAGTCCGAGGCGTTCGGCGAGTGCTTCTCCACCGAAGATCAGAAAGATGC TATGACCGCCTTCATCGAAAAGCGGAAGATCGAGGGCTTCAAGAACCGC (SEQ ID NO: 124) crotonyl-CoA ATGCATCGCACCTCGAACGGCTCCCATGCCACGGGTGGCAATCTCCCCGACGTCGCCTCGCA thioesterase TTACCCCGTGGCGTACGAGCAGACCCTGGACGGCACCGTGGGCTTCGTCATCGATGAGATG ACGCCCGAGCGTGCCACCGCCTCGGTGGAAGTTACCGACACGCTCCGCCAGCGCTGGGGCC TCGTCCACGGCGGTGCTTACTGTGCGTTGGCGGAGATGCTGGCCACGGAAGCCACGGTCGC CGTGGTGCATGAGAAGGGCATGATGGCGGTCGGCCAGTCGAATCACACCAGCTTCTTCCGC CCTGTGAAAGAGGGCCACGTGCGTGCCGAGGCCGTGCGTATTCACGCGGGCTCGACCACGT GGTTCTGGGACGTCAGCCTGCGGGACGACGCGGGTCGCCTCTGCGCCGTGTCGTCGATGTC CATCGCGGTCCGCCCTCGCCGTGAC (SEQ ID NO: 125) 4-oxalocrotonate ATGTCCACGACCAGCATCACCCCGGATGAGATCGCCCAGGTGCTGCTGGCTGGCGAGCGCA decarboxylase ACCGCACCGAGGTGGCGCAGTTCTCGGCGAGCCACCCCGACCTCGACGTCCGGACGGCCTA TGCGGCCCAGCGCGCTTTCGTCCAGGCCAAGCTGGATGCGGGCGAGCAGCTCGTCGGCTAT AAGCTGGGCCTGACCAGCCGCAACAAGCAGCGCGCCATGGGCGTCGACTGCCCGCTGTATG GCCGCGTCACGTCCTCGATGCTCGCGACGTATGGCGATCCCATCCCGTTCGACCGCTTCATCC ATCCGCGCGTCGAATCGGAGATCGCGTTCCTGCTCAAGCAGGATGTGACCGCTCCGGCGACC GTGTCGTCGGTCCTCGCGGCCACCGACGTCGTGTTCGGAGCGGTCGACGTGCTCGACTCGC GCTACGAGGGGTTCAAGTTCACGCTCGAGGATGTCGTGGCCGATAACGCGAGCGCGGGAG CGTTCTACCTCGGACCGGTCGCCCGTCCGGCCACCGAGCTCCGCCTCGACCTGCTGGGATGC ATCGTTCGCGTGGACGGCGAGGTCACCATGACCGCCGCTGGTGCGGCCGTCATGGGCCATC CCGCCGCGGCGGTCGCGTGGCTCGCCAACCAGCTCGCGCTCGAGGGCGAATCGCTGAAGGC CGGACAGCTGATCTTCTCGGGTGGCGTCACTGCGCCCGTCCCGGTCGTTCCTGGCGGCAGCG TCACGTTCGAGTTCGATGGCCTGGGCGTCATCGAGGTGGCTGGCGCC (SEQ ID NO: 126)

Production of Propylene from methane. phaC-deleted M. trichosporium transformed with a vector containing genes encoding crotonase and 4-oxalocrotonate decarboxylase are inoculated into 100 mL serum bottles containing 20-50 mL NMS media and 10 ug/mL kanamycin. The bottle headspace is flushed with a 1:1 mixture of methane and oxygen, and the bottles are sealed with butyl rubber septa and crimped. The bottles are then shaken continuously while being incubated at 30° C. The headspace gas is refreshed every 2 days as above; however, immediately prior to refreshing the headspace, samples are drawn from both the liquid phase and headspace using a 10 μL Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m×0.32 mm column and an FID maintained at 200° C. The injector is connected in splitless mode and maintained at 250° C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50° C. 1.5 min; ramp to 300° C. at 10° C./min; hold at 300° C. 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H₂O. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Note that because methane is the only carbon source provided to the cells, all propylene produced must have been derived from methane.

Example 2 Methylococcus Capsulatus Bath Strain Methanotroph

Growth and Conjugations. The procedure for conjugating plasmids from E. coli into M. capsulatus was based on the method reported in Ali, H. & Murrell, J. C. Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath. Microbiol. 155:761-771, 2009.

Briefly, a mobilizing plasmid to be conjugated was first transformed into E. coli S17-1 using standard electroporation methods. Transformation was confirmed by selection of kanamycin-resistant colonies on LB-agar containing 20 μg/mL kanamycin. Transformed colonies were inoculated into LB media containing 20 μg/mL kanamycin and shaken overnight at 37° C. A 10 mL aliquot of the overnight culture was then collected on a sterile 47 mm nitrocellulose filter (0.2 mm pore size). The E. coli donor strain was washed on the filter with 50 mL sterile NMS to remove residual media and antibiotic.

In parallel, a sample of the M. capsulatus recipient strain was inoculated into 100 mL serum bottles containing 20-50 mL NMS media. The headspace of the bottles was then flushed with a 1:1 mixture of oxygen and methane, and the bottles were sealed with butyl rubber septa and crimped. The bottles were shaken continuously in a 45° C. incubator until reaching an OD600 of approximately 0.3. The cells were then collected on the same filter as the E. coli donor strain. The filter was again washed with 50 mL of sterile NMS media. The filter was placed (cells up) on an NMS agar plate containing 0.02% (w/v) proteose peptone and incubated for 24 h at 37° C. in the presence of methane and oxygen. After 24 h, cells were re-suspended in 10 mL sterile (NMS) medium before being concentrated by centrifugation. The pellet was re-suspended in 1 mL sterile NMS media. Aliquots (100 μl ) were spread onto NMS agar plates containing 10 μg/mL kanamycin.

The plates were incubated in sealed chambers containing a 1:1 mixture of methane and oxygen maintained at 45° C. The gas mixture was replenished every 2 days until colonies formed, typically after 7-14 days. Colonies were streaked onto NMS plates containing kanamycin to confirm kanamycin resistance as well as to further isolate transformed methanotroph cells from residual E. coli donor cells.

Introduction of Propylene synthesis pathway. Note that M. capsulatus does not have a native PHA pathway, hence no pathway genes (i.e., phaC) need to be deleted. However, phaA and phaB function must be introduced to the cells to provide the substrate for the crotonase enzyme (i.e., 3-hydroxybutryl-CoA).

Selected phaA (SEQ ID NO:77), phaB (SEQ ID NO:123), crotonase (SEQ ID NO:32), crotonyl-CoA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO:10) sequences were codon optimized (see Table 5) and synthesized with appropriate promoters. The genes are then cloned and transformed into M. capsulatus as described above. Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

TABLE 5 Codon Optimized Sequences for M. capsulatus Reference Sequence Codon Optimized Sequence (SEQ ID NO: #) phaA ATGACCGACGTGGTCATCGTGTCGGCAGCCCGCACAGCAGTGGGTAAATTCGGCGGCT CGCTGGCCAAGATCGCAGCCCCGGAGCTGGGCGCCTCGGTCATCCGAGCGGTATTGGA ACGAGCCGGAGTGAAGCCCGAGCAGGTGTCGGAAGTCATCTTGGGCCAAGTGCTTACT GCCGGCAGCGGCCAAAACCCAGCCCGGCAGGCGTTGATCGCCGCAGGGCTCCCGAAC GCCGTCCCGGGCATGACGATCAACAAGGTGTGTGGCAGCGGCCTCAAGGCGGTCATGC TCGCGGCCAATGCGGTCGTAGCAGGGGACGCGGAAATCGTCGTGGCAGGCGGCCAGG AAAACATGAGCGCTGCGCCGCACGTGCTGCCCGGCTCCCGCGACGGCTTCCGGATGGG AGACGCCAAGTTGGTGGATTCAATGATCGTTGACGGGTTGTGGGACGTTTACAACAAG TACCATATGGGCATCACCGCAGAAAATGTGGCGAAAGAATATGGTATCACCCGCGAGG CCCAGGACCAGTTCGCCGCCTTGAGCCAGAACAAGGCCGAAGCTGCCCAGAAAGCGG GTCGCTTCGACGATGAAATCGTACCGATCGAAATCCCGCAACGGAAGGGCGAGCCCCT GCGCTTCGCGACCGATGAATTCGTCCGGCACGGGGTCACGGCCGAGTCCCTCGCGAGC CTCAAGCCGGCCTTCGCCAAAGAAGGCACCGTGACCGCCGCTAACGCGAGCGGCATCA ACGACGGCGCAGCCGCAGTCCTGGTTATGTCGGCGAAGAAGGCCGAAGCCCTGGGGC TGGAGCCCCTGGCCCGGATCAAGGCGTACGCCAATGCGGGAGTGGATCCGTCCGTAAT GGGAATGGGCCCTGTCCCCGCCTCGCGACGGTGCCTGGAGCGCGCAGGGTGGTCGGT AGGCGACCTCGATCTCATGGAGATCAATGAAGCGTTCGCGGCTCAGGCGCTCGCGGTG CACAAGCAGATGGGCTGGGATACCTCGAAGGTTAACGTGAACGGCGGTGCCATCGCG ATCGGCCACCCCATCGGGGCCTCAGGCTGCCGCATCCTGGTCACCCTGCTGCATGAAAT GCTGAAGCGCGATGCCAAGCGGGGACTGGCGTCGCTCTGCATCGGCGGTGGCATGGG TGTCGCCTTGGCCCTCGAGCGGCCG(SEQ ID NO: 127) pha B ATGAAGAAGGTGTGCGTCATCGGAGCCGGCACCATGGGTTCCGGGATCGCGCAGGCCT TTGCCGCCAAGGGCTTCGAAGTGGTGCTGCGTGACATCAAAGACGAGTTCGTCGACCG TGGTTTGGATTTCATCAACAAGAACCTGTCGAAACTCGTCAAGAAAGGCAAGATCGAA GAGGCAACGAAGGTTGAGATTCTCACCCGTATAAGCGGGACGGTGGACCTGAACATG GCGGCTGATTGTGACCTGGTGATCGAAGCCGCGGTGGAACGCATGGACATCAAGAAG CAGATCTTCGCAGATCTGGACAATATCTGCAAGCCAGAGACGATTCTTGCGAGCAATAC CAGCAGTCTGTCCATCACCGAGGTCGCATCCGCGACGAAACGGCCGGACAAAGTGATC GGCATGCACTTCTTCAACCCTGCGCCCGTCATGAAGTTGGTGGAAGTGATCCGGGGCAT CGCCACAAGCCAGGAAACCTTCGACGCTGTGAAAGAGACGTCGATCGCGATCGGGAAA GACCCGGTCGAGGTGGCGGAAGCACCCGGCTTCGTCGTCAATCGGATCCTGATCCCGA TGATCAATGAAGCAGTCGGCATCTTGGCCGAGGGCATTGCCAGCGTCGAAGATATCGA CAAGGCCATGAAGCTGGGCGCCAACCATCCGATGGGACCCCTGGAACTGGGAGACTTC ATCGGGCTGGACATCTGCCTGGCCATCATGGACGTTCTCTACAGCGAAACGGGCGACTC GAAGTATCGCCCGCATACCCTGCTGAAGAAATACGTCCGTGCAGGCTGGCTGGGACGC AAGTCCGGCAAGGGCTTCTACGACTATTCCAAG(SEQ ID NO: 128) crotonase ATGGAACTTAACAATGTGATCCTGGAGAAAGAAGGTAAAGTCGCCGTGGTGACCATTA ATCGCCCCAAGGCCCTGAACGCCCTGAATTCTGACACGCTGAAAGAAATGGACTACGT GATCGGCGAAATCGAGAACGACTCCGAGGTGCTGGCCGTGATCCTGACCGGCGCAGG CGAAAAGTCGTTCGTTGCCGGAGCGGATATCTCCGAGATGAAAGAGATGAACACCATT GAGGGCAGGAAGTTCGGCATCCTGGGCAATAAAGTCTTTCGCCGGCTCGAGCTCCTGG AGAAGCCGGTAATTGCCGCCGTTAATGGCTTCGCGCTCGGTGGCGGATGTGAAATCGC GATGAGCTGCGACATCCGCATAGCGAGTAGTAACGCGCGGTTCGGCCAGCCCGAGGTC GGCCTGGGCATCACGCCCGGATTCGGTGGCACTCAGCGGCTGTCGCGCCTGGTGGGCA TGGGGATGGCCAAGCAGCTGATCTTCACCGCGCAGAACATCAAAGCCGACGAAGCCCT GCGCATAGGGTTGGTGAACAAAGTCGTGGAGCCGAGCGAGTTGATGAACACCGCCAA AGAGATCGCCAACAAGATCGTCTCGAACGCACCGGTCGCGGTGAAATTGTCGAAGCAG GCCATCAACCGCGGCATGCAGTGCGATATCGATACCGCCCTCGCCTTCGAGTCGGAAGC CTTTGGTGAATGCTTCTCCACCGAAGATCAAAAAGACGCCATGACCGCCTTCATAGAGA AGCGCAAGATCGAGGGTTTTAAGAACCGG(SEQ ID NO: 129) crotonyl-CoA ATGCATCGGACCAGCAACGGCAGCCACGCCACAGGTGGCAATCTGCCGGACGTCGCTA thioesterase GCCACTATCCGGTCGCCTACGAGCAGACCCTTGATGGGACGGTGGGCTTCGTGATCGA CGAGATGACGCCAGAGCGAGCGACCGCTAGCGTCGAAGTCACCGATACGTTGCGGCA GCGGTGGGGCCTGGTCCATGGCGGTGCGTATTGCGCGCTTGCCGAAATGCTGGCCACC GAGGCTACCGTCGCCGTCGTCCACGAAAAGGGGATGATGGCGGTTGGTCAGTCGAACC ATACGTCGTTCTTTCGTCCCGTGAAAGAGGGCCACGTGCGGGCAGAAGCCGTCCGTATT CACGCCGGCAGCACCACCTGGTTCTGGGATGTTTCGCTGCGCGATGACGCCGGCAGGC TGTGCGCCGTCAGTTCCATGTCAATCGCCGTCCGTCCACGCCGGGAT (SEQ ID NO: 130) 4-oxalocrotonate ATGTCGACGACGTCCATTACCCCGGACGAGATTGCCCAGGTGCTGCTCGCTGGGGAAC decarboxylase GGAACCGCACCGAAGTGGCCCAGTTCTCCGCGTCCCATCCGGACCTGGATGTTCGCACC GCCTATGCCGCCCAGCGTGCTTTTGTCCAGGCCAAGCTGGACGCGGGAGAGCAGCTCG TCGGCTACAAGCTGGGCCTTACGAGTCGGAACAAGCAGCGTGCCATGGGTGTGGACTG CCCGCTGTACGGGCGAGTGACGAGCTCTATGCTGGCGACCTACGGGGACCCGATCCCG TTTGACCGCTTCATCCATCCGCGGGTCGAAAGCGAGATTGCGTTCCTGTTGAAACAGGA CGTGACCGCTCCGGCCACCGTGTCGTCCGTTCTGGCCGCCACGGACGTCGTCTTTGGCG CGGTCGACGTACTGGACTCCCGGTACGAAGGCTTCAAGTTCACCCTCGAAGATGTGGT GGCCGACAACGCCAGCGCTGGCGCGTTCTATCTCGGACCCGTGGCACGTCCCGCTACC GAGTTGCGCCTGGACTTGTTGGGGTGCATCGTACGTGTGGACGGCGAAGTCACGATGA CCGCGGCTGGCGCAGCCGTGATGGGCCACCCGGCAGCGGCAGTGGCCTGGCTCGCGA ACCAGCTGGCGCTGGAAGGGGAATCCCTGAAAGCCGGTCAACTGATCTTCTCGGGTGG GGTCACGGCACCCGTCCCTGTGGTGCCTGGCGGATCGGTGACCTTCGAGTTCGATGGC CTTGGCGTGATCGAGGTGGCCGGAGCA (SEQ ID NO: 131)

Production of Propylene from methane. M. capsulatus transformed with the vector described above are inoculated into 100 mL serum bottles containing 20-50 mL NMS media and 10 μg/mL kanamycin. The bottle headspace is flushed with a 1:1 mixture of methane and oxygen, and the bottles are sealed with butyl rubber septa and crimped. The bottles are then shaken continuously while being incubated at 42-45° C. The headspace gas is refreshed every 2 days as above; however, immediately prior to refreshing the headspace, samples are drawn from both the liquid phase and headspace using a 10 μL Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m×0.32 mm column and an FID maintained at 200° C. The injector is connected in splitless mode and maintained at 250° C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50° C. 1.5 min; ramp to 300° C. at 10° C./min; hold at 300° C. 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H₂O. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Note that because methane is the only carbon source provided to the cells, all propylene produced must have been derived from methane.

Example 3 Methylobacterium Extorquens Methylotroph

Growth and Transformation. The procedure for introducing plasmids into M. extorquens has been demonstrated in Ueda S., Matsumoto S., Shimizu S., and Yamane T., Transformation of a Methylotrophic Bacterium, Methylobacterium extorquens, with a Broad-Host-Range Plasmid by Electroporation, Appl. Environ. Microbiol., 1991, April; 57(4): 924-926.

Briefly, wild-type (wt) M. extorquens is cultured at 30° C. in NMS media supplemented with 1% methanol. Cells of M. extorquens NR-2 grown to the middle logarithmic phase (1.4×10⁹/ml) are harvested by centrifugation at 6,000×g for 10 min and washed with electroporation buffer (10 mM Tris-HCl, 2 mM MgCl₂.6H₂O, 10% [wt/vol] sucrose [pH 7.5]). Cells are re-suspended in the same buffer at a cell concentration of 7.0×10¹⁰/ml. The cell suspension and the solution of vector (70 μg/mL) are mixed at a ratio of 9:1 (vol/vol) in an Eppendorf tube. The mixture (10 μL) is then transferred into a space between the electrodes of a chamber, where it is equilibrated for 3 min. After being subjected to 10 pulses of a 10 kV/cm electric field for 300 μsec/pulse, a 5 μL aliquot of the mixture is transferred to an Eppendorf tube. 0.2 mL of NMS medium is then added to the tube. The cell suspension is then incubated for 2 h at 30° C. to allow expression of the antibiotic resistance genes prior to plating on NMS plates containing 1% methanol and 20 μg/mL kanamycin.

The plates were incubated at 30° C. until colonies formed. Colonies were streaked onto duplicate plates to confirm kanamycin resistance as well as to further isolate transformed methylotroph cells from residual E. coli donor cells.

Deletion of phaC. The deletion of the phaC gene has been described in Korotkova N., Lidstrom M. E., Connection between poly-beta-hydroxybutyrate biosynthesis and growth on C(1) and C(2) compounds in the methylotroph Methylobacterium extorquens AM1, J. Bacteriol. 2001 February; 183(3):1038-46.

Briefly, insertion cassettes containing a kanamycin resistance marker were constructed with flanking sequences homologous to the areas flanking the phaC gene in the M. extorquens genome. A tetracycline resistance gene was incorporated elsewhere in the plasmid. Transformants were initially selected for resistance to kanamycin, and then screened for sensitivity to tetracycline to identify potential double cross-over recombination events. Correct insertion into and deletion of the phaC gene was confirmed by PCR.

Loss of phaC function is confirmed by growing the cells under nitrogen limited conditions and measuring PHA content as described in Pieja A J, Rostkowski K H, Criddle C S, Distribution and selection of poly-3-hydroxybutyrate production capacity in methanotrophic proteobacteria, Microb. Ecol. 2011 October; 62(3):564-73.

Introduction of Propylene synthesis pathway. Selected crotonase (SEQ ID NO:32), crotonyl-coA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO:10) sequences were codon optimized (see Table 6) and synthesized with appropriate promoters. The genes are then cloned and transformed into the phaC knockout strain as described above. Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

TABLE 6 Codon Optimized Sequences for M. extorquens Reference Sequence Codon Optimized Sequence (SEQ ID NO: #) crotonase ATGGAGCTGAACAACGTCATCCTCGAAAAAGAGGGCAAGGTGGCGGTCGTCACCATC AACCGCCCCAAGGCCCTCAACGCGCTCAACAGCGACACGCTCAAAGAAATGGATTAC GTCATCGGCGAGATCGAGAACGATTCCGAGGTGCTCGCCGTGATCCTCACCGGTGCG GGCGAAAAGTCGTTCGTGGCGGGTGCGGATATCTCCGAAATGAAAGAAATGAACACG ATCGAGGGCCGGAAGTTCGGCATCCTCGGCAACAAGGTTTTCCGCCGTCTCGAGTTGT TGGAGAAGCCGGTCATTGCCGCCGTGAATGGCTTCGCCCTCGGTGGTGGCTGCGAGA TCGCCATGAGCTGCGACATCCGGATCGCGTCGAGCAACGCCCGTTTCGGCCAGCCGG AAGTCGGCTTGGGCATCACCCCGGGCTTCGGCGGCACGCAGCGCCTCTCGCGGCTCG TCGGCATGGGCATGGCCAAGCAGCTCATCTTCACCGCCCAGAATATCAAGGCGGACG AGGCGCTGCGCATTGGCCTCGTTAACAAGGTCGTGGAGCCCTCGGAGCTCATGAACA CCGCGAAAGAGATCGCGAACAAGATCGTGTCCAACGCACCGGTGGCCGTCAAGCTCT CGAAGCAGGCCATCAACCGCGGCATGCAGTGCGATATCGACACCGCGCTCGCGTTCG AGAGCGAGGCGTTCGGGGAGTGCTTCTCGACCGAAGATCAGAAGGACGCCATGACC GCCTTCATCGAGAAGCGCAAGATCGAAGGCTTCAAGAACCGC (SEQ ID NO: 132) crotonyl-coA ATGCACCGCACCTCGAACGGCTCGCACGCCACCGGTGGCAACCTGCCGGACGTCGCCT thioesterase CGCATTACCCGGTCGCGTACGAACAGACCCTGGACGGGACGGTGGGCTTCGTCATCG ATGAGATGACGCCCGAGCGCGCGACGGCCTCGGTCGAGGTGACCGACACGCTCCGCC AGCGCTGGGGCCTCGTCCACGGCGGTGCGTACTGCGCCCTCGCCGAGATGCTCGCCA CCGAGGCGACGGTGGCCGTGGTCCATGAGAAGGGCATGATGGCGGTGGGGCAGAGC AACCACACGAGCTTCTTTCGCCCGGTGAAAGAGGGCCACGTCCGCGCAGAGGCCGTG CGCATCCACGCGGGCTCCACCACCTGGTTTTGGGATGTGTCGCTGCGCGATGACGCAG GCCGCCTTTGCGCCGTGTCCAGCATGTCGATCGCGGTGCGGCCCCGCCGCGAC (SEQ ID NO: 133) 4-oxa locrotonate ATGAGCACCACGTCGATCACCCCGGACGAGATCGCGCAGGTGCTGCTGGCAGGCGAG decarboxylase CGCAACCGGACCGAGGTCGCCCAGTTCAGCGCCTCGCACCCGGACCTCGACGTGCGC ACGGCGTATGCTGCGCAGCGGGCGTTCGTGCAGGCCAAGCTCGATGCCGGCGAGCA GTTGGTCGGCTACAAGCTCGGCCTGACCTCGCGGAATAAGCAGCGGGCCATGGGCGT CGACTGCCCGTTGTATGGTCGCGTCACCAGCAGCATGCTGGCGACCTACGGCGACCCC ATCCCCTTCGACCGCTTCATCCATCCGCGCGTCGAATCGGAAATCGCCTTCCTGCTGAA GCAGGATGTCACCGCCCCGGCCACCGTCTCGTCGGTCCTCGCCGCGACCGACGTCGTT TTCGGCGCTGTCGACGTGCTGGATAGCCGCTACGAGGGCTTCAAGTTCACGCTGGAA GATGTGGTCGCGGACAACGCCAGCGCCGGAGCCTTCTACCTCGGTCCCGTCGCCCGTC CGGCCACGGAGCTCCGGCTCGACTTGCTCGGCTGCATCGTCCGGGTCGACGGCGAGG TTACCATGACCGCAGCGGGAGCCGCCGTGATGGGCCACCCCGCAGCCGCGGTGGCCT GGCTCGCCAACCAGCTCGCCCTCGAGGGCGAGTCGCTGAAAGCCGGCCAGCTGATCT TCAGCGGCGGTGTGACGGCGCCGGTCCCCGTCGTGCCCGGTGGCTCGGTCACCTTCG AGTTCGACGGACTGGGCGTCATCGAGGTGGCCGGCGCC (SEQ ID NO: 134)

Production of Propylene from methanol. phaC-deleted M. extorquens transformed with a vector containing genes encoding crotonase and 4-oxalocrotonate decarboxylase are inoculated into 100 mL sealed flasks containing 20-50 mL NMS media, 125 mM methanol, 50 μg/mL rifamycin, and 10 μg/mL kanamycin. The flask headspace is flushed with oxygen and sealed to prevent loss of the propylene product. The flasks are then shaken continuously while being incubated at 30° C. The headspace gas is refreshed every day; however, immediately prior to refreshing the headspace, samples are drawn from both the liquid phase and headspace using a 10 μL Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m x 0.32 mm column and an FID maintained at 200° C. The injector is connected in splitless mode and maintained at 250° C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50° C. 1.5 min; ramp to 300° C. at 10° C./min; hold at 300° C. 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H₂O. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Note that because methanol is the only carbon source provided to the cells, all propylene produced must have been derived from methanol.

Example 4 Clostridiuma Utoethanogenum

Growth and transformation. C. autoethanogenum is cultivated anaerobically in modified PETC medium (ATCC medium 1754) at 37° C. in modified PETC media.

The modified PETC medium contains (per L) 1 g NH₄Cl, 0.4 g KCl, 0.2 g MgSO₄×7H₂O, 0.8 g NaCl, 0.1 g KH₂PO₄, 20 mg CaCl₂×2H₂O, 10 ml trace elements solution (see below), 10 ml Wolfe's vitamin solution (see below), 2 g NaHCO₃, and 1 mg resazurin. After the pH is adjusted to 5.6, the medium is boiled, dispensed anaerobically, and autoclaved at 121° C. for 15 min. Steel mill waste gas (composition: 44% CO, 32% N₂, 22% CO₂, 2% H₂) or equivalent synthetic mixtures are used as carbon source. The media has a final pH of 5.9 and is reduced with Cystein-HCl and Na₂S in a concentration of 0.008% (w/v).

The trace elements solution consists of 2 g nitrilotriacetic acid (adjusted to pH 6 with KOH before addition of the remaining ingredients), 1 g MnSO₄, 0.8 g Fe(SO₄)₂(NH₄)₂×6H₂O, 0.2 g CoCl₂×6H₂O, 0.2 mg ZnSO₄×7H₂O, 20 mg CuCl₂×2H₂O, 20 mg NiCl₂×6H₂O, 20 mg Na₂MoO₄×2H₂O, 20 mg Na₂SeO₄, and 20 mg Na₂WO₄ per liter.

Wolfe's vitamin solution (Wolin et al., 1963, J. Biol. Chem. 238:2882-2886) contains (per L) 2 mg biotin, 2 mg folic acid, 10 mg pyridoxine hydrochloride, 5 mg thiamine-HCl, 5 mg riboflavin, 5 mg nicotinic acid, 5 mg calcium D-(+)-pantothenate, 0.1 mg vitamin B12, 5 mg p-aminobenzoic acid, and 5 mg thioctic acid.

Growth experiments are carried out in a volume of 100 ml PETC media in plastic-coated 500-ml-Schott Duran® GL45 bottles with butyl rubber stoppers and 200 kPa steel mill waste gas as sole energy and carbon source. Growth is monitored by measuring the optical density at 600 nm (OD600 nm).

Transformation methods for C. autoethanogenum are performed as described in U.S. Patent Publication 2011/0236941.

Briefly, to make competent cells, a 50 ml culture of C. autoethanogenum is subcultured to fresh media for 3 consecutive days. These cells are used to inoculate 50 ml PETC media containing 40 mM DL-threonine at an OD600 nm of 0.05. When the culture reaches an OD600 nm of 0.4, the cells are transferred into an anaerobic chamber and harvested at 4,700×g and 4° C. The culture is twice washed with ice-cold electroporation buffer (270 mM sucrose, 1 mM MgCl₂, 7 mM sodium phosphate, pH 7.4) and finally suspended in a volume of 600 μl fresh electroporation buffer. This mixture is transferred into a pre-cooled electroporation cuvette with a 0.4 cm electrode gap containing 1 μg of the methylated plasmid mix and immediately pulsed using the Gene pulser Xcell electroporation system (Bio-Rad) with the following settings: 2.5 kV, 600 μl, and 25 μF. Time constants of 3.7-4.0 ms are achieved. The culture is transferred into 5 ml fresh media. Regeneration of the cells is monitored at a wavelength of 600 nm using a Spectronic Helios Epsilon Spectrophotometer (Thermo) equipped with a tube holder. After an initial drop in biomass, the cells start growing again. Once the biomass has doubled from that point, the cells are harvested, suspended in 200 μl fresh media and plated on selective PETC plates (containing 1.2% Bacto™ Agar (BD)) with 4 μg/ μl Clarithromycin. After 4-5 days of incubation with 30 psi steel mill gas at 37° C., 15-80 colonies per plate are clearly visible.

The colonies are used to inoculate 2 ml PETC media containing 4 μg/ μl Clarithromycin. When growth occurs, the culture is upscaled into 5 ml and later 50 ml PETC media containing 4 μg/ μl Clarithromycin and 30 psi steel mill gas as sole carbon source.

Confirmation of Successful Transformation:

To verify the DNA transfer, a plasmid mini prep is performed from 10 ml culture volume using the QIAprep Spin Miniprep Kit (Qiagen). The quality of the isolated plasmid DNA is sufficient to run a control PCR. The PCR is performed with Illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) using standard conditions (95° C. for 5 min; 32 cycles of 95° C. for 30 s, 50° C. for 30 s, and 72° C. for 1 min; 72° C. for 10 min). As a further control, 1 μl of each of the partly degraded isolated plasmids are re-transformed in E. coli XL1-Blue MRF′ Kan (Stratagene), from where the plasmids can be isolated cleanly and verified by restriction digests.

Introduction of Propylene synthesis pathway. Note that C. autoethanogenum does not have a native PHA pathway, hence no pathway genes need to be deleted. However, phaA and phaB function must be introduced to the cells to provide the substrate for the crotonase enzyme.

Selected phaA (SEQ ID NO:77), phaB (SEQ ID NO:123), crotonase (SEQ ID NO:32), crotonyl-coA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO:10) sequences were codon optimized (see Table 7) and synthesized with appropriate promoters. The genes are then cloned and transformed into C. autoethanogenum as described above. Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

TABLE 7 Codon Optimized Sequences for C. autoethanogenum Reference Sequence Codon Optimized Sequence (SEQ ID NO: #) phaA ATGACAGATGTAGTGATAGTTTCAGCAGCTAGAACAGCTGTTGGTAAATTCGGTGGTTC GTTAGCGAAAATAGCTGCTCCTGAATTAGGAGCTTCAGTAATTAGAGCTGTATTAGAAA GAGCAGGTGTAAAACCTGAGCAAGTGTCTGAAGTCATATTAGGGCAAGTCTTGACTGCA GGGTCAGGTCAGAATCCTGCAAGACAAGCCTTAATAGCTGCGGGACTTCCTAATGCAGT ACCTGGGATGACAATCAATAAAGTTTGTGGATCAGGTCTAAAAGCAGTTATGTTGGCTG CAAATGCGGTTGTAGCTGGAGACGCTGAAATAGTTGTGGCGGGTGGACAAGAAAACAT GAGTGCAGCACCACATGTTCTACCTGGCAGTAGAGATGGATTTCGAATGGGAGATGCAA AGCTAGTAGATAGCATGATAGTAGATGGATTATGGGATGTTTACAATAAGTATCATATG GGAATAACTGCAGAAAATGTAGCAAAAGAATATGGAATTACACGTGAAGCTCAAGACCA ATTTGCAGCACTTTCACAGAATAAGGCTGAAGCAGCACAAAAAGCTGGAAGATTTGATG ATGAAATAGTTCCTATTGAAATTCCACAAAGAAAGGGAGAACCACTTAGATTTGCCACTG ATGAATTTGTAAGGCATGGAGTAACAGCTGAATCTCTTGCAAGTTTGAAACCAGCGTTTG CCAAAGAGGGAACTGTGACTGCTGCTAATGCTTCAGGCATAAATGATGGAGCTGCAGCA GTCCTTGTTATGTCTGCGAAGAAAGCAGAAGCTCTTGGCCTTGAACCTTTGGCACGTATT AAGGCTTATGCCAATGCTGGAGTTGATCCTTCTGTTATGGGAATGGGACCTGTACCGGCA AGTAGAAGATGCCTAGAAAGAGCAGGATGGAGTGTAGGTGATTTAGATCTTATGGAGA TTAATGAGGCTTTTGCTGCACAAGCGTTGGCTGTTCATAAGCAAATGGGTTGGGATACAT CAAAAGTTAATGTAAATGGCGGTGCAATAGCAATTGGACATCCAATAGGAGCATCTGGT TGCAGAATACTTGTTACTCTTCTTCATGAAATGTTGAAAAGAGATGCTAAAAGAGGTTTA GCATCATTATGTATAGGTGGTGGCATGGGAGTAGCTTTAGCATTAGAAAGACCG(SEQ ID NO: 135) pha B ATGAAAAAGGTTTGTGTTATAGGTGCAGGTACTATGGGTTCAGGTATTGCTCAGGCATTT GCAGCCAAAGGGTTTGAAGTTGTTTTAAGGGACATAAAAGATGAATTCGTGGATAGGG GATTAGATTTTATAAATAAGAACTTAAGTAAGCTTGTAAAGAAGGGCAAAATTGAAGAG GCTACTAAAGTAGAAATCTTGACGAGAATAAGTGGTACCGTAGATCTTAACATGGCTGC AGATTGTGATTTAGTTATTGAAGCTGCGGTCGAAAGAATGGACATTAAGAAACAGATTTT TGCAGACTTAGATAACATATGTAAGCCAGAAACTATCTTAGCCAGTAATACAAGCTCATT ATCAATTACTGAAGTAGCAAGTGCGACAAAAAGGCCTGATAAAGTAATTGGAATGCATT TCTTTAATCCAGCACCTGTTATGAAATTAGTGGAAGTTATAAGGGGAATAGCAACTTCAC AAGAAACTTTTGATGCAGTGAAAGAAACCTCAATTGCAATAGGTAAAGACCCCGTTGAA GTTGCTGAAGCACCAGGTTTTGTTGTTAATAGAATACTAATACCAATGATAAATGAAGCA GTTGGAATCCTTGCAGAAGGTATAGCAAGTGTAGAAGATATTGACAAAGCAATGAAATT AGGTGCAAACCATCCAATGGGTCCTTTGGAATTAGGAGATTTCATTGGATTAGATATATG TTTAGCAATAATGGATGTACTATATTCTGAGACTGGAGATTCTAAGTACAGGCCTCATAC TTTACTTAAGAAATATGTAAGGGCGGGATGGTTAGGAAGAAAGTCTGGAAAGGGCTTTT ATGATTATAGTAAG(SEQ ID NO: 136) crotonase ATGGAACTTAACAATGTAATACTTGAAAAAGAAGGCAAAGTAGCTGTTGTAACAATAAA CAGGCCAAAAGCTCTAAATGCACTTAATTCCGACACTCTTAAAGAAATGGATTACGTTAT AGGTGAGATAGAAAATGATTCTGAAGTACTAGCTGTAATACTTACAGGTGCTGGTGAGA AATCATTTGTGGCAGGAGCAGATATTTCTGAAATGAAAGAAATGAATACTATTGAGGGG AGAAAATTCGGGATACTTGGAAACAAGGTTTTTAGAAGGTTAGAATTACTTGAGAAACC AGTAATAGCTGCCGTAAATGGATTTGCATTAGGTGGCGGATGTGAAATAGCAATGTCAT GCGATATCCGAATCGCATCTTCTAATGCAAGATTTGGGCAACCTGAAGTTGGATTAGGAA TCACTCCCGGATTTGGCGGTACACAAAGACTTAGCAGATTAGTAGGTATGGGAATGGCT AAGCAACTAATTTTTACGGCTCAGAACATAAAAGCAGATGAAGCTCTTAGGATTGGACTT GTGAATAAAGTAGTAGAACCGTCGGAGCTTATGAATACAGCAAAAGAAATTGCAAACAA AATAGTAAGTAATGCACCAGTGGCAGTTAAACTTTCGAAACAAGCAATCAATAGGGGCA TGCAATGCGATATAGATACGGCTTTGGCATTTGAAAGTGAAGCATTTGGGGAATGTTTTT CAACGGAAGATCAAAAAGATGCTATGACAGCCTTTATTGAGAAAAGAAAGATAGAGGG ATTTAAGAATAGA (SEQ ID NO: 137) crotonyl-coA ATGCACAGAACATCTAATGGATCACATGCAACAGGTGGCAATCTACCAGATGTTGCAAG thioesterase TCATTATCCGGTAGCTTATGAACAGACATTAGATGGAACCGTTGGTTTTGTGATAGATGA AATGACTCCAGAAAGAGCTACAGCTTCCGTCGAGGTAACTGATACATTACGTCAGAGGT GGGGTTTGGTTCATGGTGGAGCATATTGTGCTCTTGCGGAAATGTTGGCTACTGAAGCA ACAGTTGCAGTTGTACATGAAAAAGGTATGATGGCAGTTGGTCAATCTAATCACACCAG CTTTTTCAGGCCAGTTAAAGAAGGTCATGTTAGAGCCGAGGCGGTTAGGATACATGCAG GAAGTACAACCTGGTTTTGGGATGTTTCTTTAAGAGATGATGCTGGTAGATTATGTGCTG TTAGCAGTATGTCCATTGCAGTAAGACCAAGAAGAGAT (SEQ ID NO: 138) 4-oxalocrotonate ATGAGCACTACTAGTATAACACCAGATGAAATTGCTCAAGTACTATTAGCTGGAGAAAG decarboxylase AAATAGAACAGAAGTAGCACAGTTTTCAGCTTCACACCCGGATTTAGATGTAAGAACGG CTTATGCTGCTCAAAGAGCATTTGTTCAAGCAAAACTTGATGCAGGAGAGCAGTTAGTA GGCTATAAGCTTGGACTTACATCTAGGAATAAACAAAGAGCTATGGGTGTAGATTGCCC ACTTTATGGAAGAGTTACGTCCTCTATGTTGGCCACATATGGAGATCCAATACCATTCGA CAGATTCATACATCCTAGAGTTGAGTCTGAAATTGCATTCTTATTGAAACAAGATGTTACT GCTCCTGCTACAGTATCATCCGTACTTGCTGCAACTGATGTAGTTTTTGGTGCAGTGGAT GTTTTGGATTCAAGATATGAAGGATTTAAGTTTACTCTAGAAGATGTAGTTGCAGATAAT GCCAGTGCAGGAGCTTTTTACCTTGGACCTGTTGCTAGACCTGCTACAGAGTTAAGACTT GATTTACTAGGATGTATAGTTAGAGTTGACGGAGAAGTTACAATGACAGCGGCTGGTGC CGCTGTTATGGGACACCCTGCTGCTGCTGTAGCATGGTTAGCTAATCAACTTGCACTTGA GGGTGAAAGCTTGAAGGCAGGTCAGCTTATCTTTAGCGGTGGGGTCACTGCTCCTGTTC CAGTAGTTCCTGGTGGAAGCGTGACCTTTGAATTTGATGGCCTAGGTGTAATAGAAGTA GCAGGAGCC (SEQ ID NO: 139)

Production of Propylene from carbon monoxide. C. ethanogenum transformed with the vector described above are used to inoculate 2 ml PETC media containing 4 μg/ μl Clarithromycin. When growth occurs, the culture is upscaled into 5 ml and later 50 ml PETC media containing 4 μg/ μl Clarithromycin and 30 psi steel mill gas as sole carbon source. The bottles are then shaken continuously while being incubated at 37° C. The headspace gas is refreshed every 2 days; however, immediately prior to refreshing the headspace, samples are drawn from both the liquid phase and headspace using a 10 μL Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m×0.32 mm column and an FID maintained at 200° C. The injector is connected in splitless mode and maintained at 250° C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50° C. 1.5 min; ramp to 300° C. at 10° C./min; hold at 300° C. 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H₂O. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Note that because carbon monoxide is the only carbon source provided to the cells, all propylene produced must have been derived from carbon monoxide.

Example 5 Escherichia Coli Heterotroph

Growth and Transformation. Growth and transformation methods for E. coli are well-known in the art. E. coli strains were transformed by electroporation using the appropriate plasmids. A single colony from a fresh transformation was then used to seed an overnight culture grown in Luria Broth (LB) supplemented with 1.5% (w/v) glucose and appropriate antibiotics at 37° C. in a rotary shaker (200 r.p.m.). Antibiotics were used at a concentration of 50 μg/ml for strains with a single resistance marker. For strains with multiple resistance markers, kanamycin and chloramphenicol were used at 25 μg/ml and carbenicillin was used at 50 μg/ml.

Cloning and expression of 4-OD genes. 4-OD genes were identified from BLAST searching of the NCBI database using the Pseudomonas putida 4-OD sequence as a starting sequence. 24 individual 4-OD proteins were chosen for expression studies. The proteins were reverse-translated and codon-optimized for E. coli using commercial methods (DNA2.0, Inc. Menlo Park, Calif.). The genes were then cloned under control of a T7 promoter and expressed in E. coli BL21 (DE3). Briefly, single colonies were inoculated into 2 mL cultures of LB containing 50 μg/mL kanamycin and shaken overnight at 37° C. 1 mL of the saturated overnight culture was then inoculated into 200 mL LB containing 50 μg/mL kanamycin in a 2 L flask. The flasks were then shaken at 37° C. for 3-4 hours until an OD600 of 0.5-1.0 was reached. The cultures were induced by addition of 1 mM IPTG and shaken for additional 3 h at 37° C. Cells were harvested by centrifugation and analyzed by SDS-PAGE to confirm protein expression (see FIG. 5). Activity in cell lysates was confirmed by measuring decarboxylation activity on 4-oxalocrotonate (see FIG. 6).

4-OD Lysate Preparation

Lysates were generated by resuspending induced E. coli cell pellet (equivalent to 1 mL culture) in 0.5 mL lysis buffer (20 mM KHPO₄ pH 8.0; 0.3 M KCl; 10% (w/v) glycerol; 0.1% NP-40; 0.5 mg/ml lysozyme; 1 mM PMSF). Cells were sonicated 5 seconds and then centrifuged for 15 min at 15,000×g, 4° C. The cleared supernatant was assayed immediately or stored at −80° C. for later assay.

4-Oxalocrotonate Decarboxylation Activity Assay

The assay buffer comprised 100 mM Tris-HCl pH 7.4; 3.3 mM MgSO₄; 1 mM 4-oxalocrotonate (the stock solution of 4-oxalocrotonate was pre-equilibrated with a 1:100 dilution of E. coli lysate expressing 4-oxalocrotonate tautomerase from Pseudomonas putida (UniProtKB/Swiss-Prot Accession No. Q01468, geneid 87856) to achieve a distribution of keto and enol forms of 4-oxalocrotonate). Total reaction volume was 200 μl in a 96 well UV transparent plate and read on a SpectraMax Plate Reader (Molecular Devices). The reaction was initiated by addition of 4-OD containing lysates at 1:10 to 1:1000 final dilutions. Reactions were run at 25° C. Consumption of substrate was monitored by measuring drop in absorbance at 240 nm for the keto tautomer and at 300 nm for the enol tautomer (see FIG. 6).

Crotonate Decarboxylation Assay

The assay buffer comprised 100 mM Tris-HCl pH 7.4; 3.3 mM MgSO₄; 87.2 mM crotonic acid. Reactions were initiated by adding lysates to a final concentration of 1:10 to 1:100 in 1 ml volume in a TargetDP vial (2 ml total volume). Reactions were incubated from 12 to 72 hours at room temperature. Generated propylene was detected by injection of 0.5 μl aqueous phase plus 2 μl headspace gas onto a HP5890 GC equipped with a CP-PoraBOND U 25 m×0.32 mm column and an FID maintained at 200° C. The injector was connected in splitless mode and maintained at 250° C. Samples were run with He Gas at 7.3 ml/min as a carrier gas; the oven program was set as follows: hold at 50° C. 1.5 min; ramp to 300° C. at 10° C./min; hold at 300° C. 10 min. Propylene was identified by retention time compared to pure propylene diluted in air or dissolved in pure H₂O (see FIG. 7).

Introduction of Propylene Synthesis Pathway

Introduction of the pathway is performed essentially as described in Bond-Watts et al., Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat. Chem. Biol. 7: 222-227, 2011.

Selected phaA (SEQ ID NO:77), phaB (SEQ ID NO:123), crotonase (SEQ ID NO:32), crotonyl-coA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO:10) sequences were codon optimized (see Table 8) and synthesized with appropriate promoters. The genes are then cloned and transformed into E. coli strain BL21 (DE3). Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

TABLE 8 Codon Optimized Sequences for E. coli Reference Sequence Codon Optimized Sequence (SEQ ID NO: #) phaA ATGACCGACGTGGTGATCGTGAGCGCTGCGCGCACGGCGGTTGGCAAGTTTGGTGGTA GCCTGGCGAAGATCGCGGCACCGGAGTTGGGCGCCAGCGTTATTCGTGCCGTCCTGGAA CGCGCAGGTGTGAAACCGGAGCAGGTGAGCGAAGTGATCCTGGGTCAAGTGCTGACCG CAGGCAGCGGTCAAAACCCGGCACGTCAAGCCTTGATTGCCGCAGGTCTGCCAAACGCT GTTCCGGGCATGACCATTAACAAAGTGTGTGGTTCTGGTCTGAAAGCGGTGATGCTGGC TGCGAACGCGGTTGTCGCCGGTGATGCGGAAATTGTGGTCGCGGGTGGCCAGGAGAAT ATGTCCGCAGCTCCGCACGTGCTGCCGGGCAGCCGTGACGGTTTCCGTATGGGCGATGC TAAATTGGTAGATAGCATGATTGTTGACGGCTTGTGGGACGTGTATAACAAATATCACAT GGGTATCACCGCGGAAAACGTTGCGAAAGAGTACGGTATCACCCGTGAGGCGCAGGAC CAGTTTGCCGCACTGAGCCAGAACAAGGCCGAAGCGGCGCAAAAAGCAGGCCGTTTTG ATGATGAGATCGTTCCGATTGAGATTCCGCAGCGTAAAGGTGAACCGCTGCGCTTCGCT ACCGACGAGTTTGTCCGTCACGGCGTTACCGCCGAATCCCTGGCCTCTTTGAAACCGGCG TTTGCTAAAGAGGGTACCGTCACCGCGGCAAACGCAAGCGGTATTAACGATGGCGCAGC AGCTGTCCTGGTTATGTCCGCGAAGAAGGCAGAAGCGTTGGGCCTGGAGCCGCTGGCTC GCATTAAAGCATATGCCAATGCCGGCGTTGATCCGAGCGTTATGGGCATGGGTCCGGTC CCGGCAAGCCGTCGTTGCCTGGAGCGTGCAGGCTGGTCCGTTGGCGACCTGGATCTGAT GGAGATCAATGAAGCCTTCGCAGCGCAGGCGCTGGCAGTGCACAAGCAGATGGGTTGG GACACCAGCAAGGTTAATGTCAATGGTGGCGCAATCGCCATTGGCCATCCTATCGGTGC GAGCGGTTGTCGTATTTTGGTTACCCTGCTGCATGAAATGCTGAAACGCGACGCCAAGC GTGGCCTGGCTAGCCTGTGCATCGGTGGTGGTATGGGTGTGGCGCTGGCGCTGGAACG TCCA (SEQ ID NO: 140) pha B ATGAAGAAAGTATGCGTCATCGGTGCGGGCACCATGGGCAGCGGTATTGCGCAGGCGT TTGCAGCCAAGGGCTTCGAGGTGGTCCTGCGCGATATCAAAGATGAGTTCGTTGATCGC GGTTTGGACTTCATCAACAAAAACCTGAGCAAGCTGGTTAAGAAGGGTAAGATCGAAGA GGCGACGAAGGTTGAAATTCTGACCCGCATCAGCGGTACTGTTGACCTGAATATGGCGG CAGACTGCGATTTGGTTATTGAAGCTGCGGTCGAGCGTATGGACATTAAGAAGCAGATT TTCGCCGATCTGGACAACATTTGTAAGCCGGAGACGATTCTGGCGAGCAACACCAGCAG CTTGAGCATTACCGAGGTGGCCTCTGCCACGAAGCGTCCGGATAAGGTCATCGGTATGC ACTTCTTTAACCCGGCTCCGGTGATGAAACTGGTCGAGGTGATCCGCGGTATTGCTACCA GCCAAGAAACGTTTGACGCTGTGAAAGAGACGTCGATCGCTATCGGCAAGGATCCGGTT GAGGTGGCAGAAGCTCCGGGTTTTGTGGTGAATCGCATCCTGATCCCGATGATCAACGA GGCCGTAGGTATCCTGGCCGAGGGTATTGCCTCTGTGGAAGATATCGACAAGGCGATGA AACTGGGTGCTAATCACCCGATGGGTCCGTTGGAGCTGGGTGACTTCATCGGTCTGGAC ATTTGTCTGGCGATCATGGACGTTCTGTACTCTGAGACGGGCGACAGCAAATATCGCCCG CACACCCTGCTGAAAAAGTACGTTCGTGCTGGTTGGCTGGGTCGTAAGTCTGGCAAAGG CTTCTACGATTACAGCAAG(SEQ ID NO: 141) crotonase ATGGAGCTGAATAATGTGATTCTGGAGAAAGAGGGCAAAGTCGCTGTTGTTACGATTAA CCGCCCGAAGGCATTGAACGCCCTGAACAGCGATACCCTGAAAGAGATGGATTACGTGA TTGGCGAGATCGAAAACGACAGCGAAGTTCTGGCCGTCATTCTGACTGGTGCCGGTGAA AAGAGCTTTGTCGCGGGTGCAGATATTAGCGAGATGAAAGAGATGAATACGATCGAAG GTCGTAAATTCGGTATCCTGGGCAATAAAGTCTTTCGTCGTTTGGAACTGCTGGAGAAAC CTGTCATCGCTGCCGTGAATGGCTTCGCGCTGGGCGGTGGCTGCGAGATTGCAATGAGC TGCGATATCCGTATCGCGAGCAGCAATGCGCGTTTCGGTCAACCGGAAGTGGGTCTGGG TATCACGCCGGGTTTTGGTGGCACCCAACGCCTGAGCCGTTTGGTTGGCATGGGTATGG CAAAACAACTGATCTTTACCGCGCAGAACATCAAAGCAGATGAAGCTCTGCGCATTGGCT TGGTCAATAAGGTGGTTGAGCCGAGCGAACTGATGAACACGGCGAAAGAGATCGCGAA CAAGATCGTGAGCAATGCACCGGTGGCCGTCAAACTGAGCAAACAGGCCATCAATCGTG GTATGCAATGTGATATCGACACCGCGCTGGCATTCGAAAGCGAGGCATTTGGTGAGTGC TTCAGCACGGAAGATCAAAAGGATGCAATGACGGCGTTCATTGAAAAACGTAAGATTGA AGGCTTCAAGAACCGC (SEQ ID NO: 142) crotonyl-coA ATGCATCGTACGAGCAACGGCAGCCACGCCACCGGTGGCAATCTGCCTGATGTCGCGAG thioesterase CCATTATCCGGTTGCATACGAACAGACCCTGGACGGCACCGTCGGTTTCGTGATTGACGA AATGACTCCGGAGCGTGCCACCGCGAGCGTTGAGGTCACCGATACCCTGCGCCAGCGTT GGGGTCTGGTTCATGGTGGTGCATATTGTGCGTTGGCAGAGATGTTGGCGACTGAAGCG ACCGTCGCAGTAGTCCATGAAAAGGGCATGATGGCGGTGGGCCAAAGCAATCACACCA GCTTTTTCCGTCCGGTTAAAGAGGGCCATGTTCGCGCAGAGGCGGTGCGTATTCACGCG GGTAGCACGACCTGGTTCTGGGACGTTAGCCTGCGCGATGACGCAGGTCGTCTGTGTGC AGTTAGCAGCATGTCTATTGCTGTCCGTCCGCGTCGCGAC (SEQ ID NO: 143) 4-oxa locrotonate ATGAGCACCACGAGCATTACCCCGGACGAAATCGCGCAGGTTCTGCTGGCAGGCGAGC decarboxylase GTAATCGCACCGAGGTGGCGCAATTTTCTGCGTCGCACCCGGATTTGGATGTCCGTACCG CGTACGCGGCACAACGTGCGTTCGTTCAGGCTAAACTGGACGCAGGTGAACAACTGGTC GGTTACAAATTGGGTCTGACGTCTCGTAATAAGCAGCGCGCAATGGGTGTCGACTGCCC GCTGTACGGCCGTGTTACCAGCAGCATGCTGGCCACCTATGGTGATCCGATTCCGTTTGA CCGCTTTATTCACCCACGTGTGGAGTCCGAAATTGCGTTCCTGCTGAAGCAAGATGTGAC CGCACCGGCGACGGTCAGCAGCGTTCTGGCAGCGACTGACGTCGTGTTTGGCGCGGTTG ACGTCCTGGATAGCCGTTACGAGGGCTTCAAGTTCACCCTGGAAGATGTTGTTGCAGAC AATGCATCTGCGGGTGCTTTCTATCTGGGTCCAGTTGCACGTCCAGCGACGGAGCTGCGT CTGGATCTGCTGGGTTGCATTGTGCGCGTCGACGGCGAAGTGACCATGACCGCAGCGGG TGCCGCGGTTATGGGCCACCCGGCAGCGGCCGTTGCGTGGCTGGCCAATCAGCTGGCCC TGGAAGGTGAAAGCCTGAAGGCGGGTCAGCTGATCTTCAGCGGTGGTGTCACTGCGCC GGTCCCGGTTGTGCCGGGTGGCAGCGTGACCTTCGAGTTTGACGGCCTGGGTGTCATCG AAGTGGCAGGTGCA (SEQ ID NO: 144)

Production of Propylene from glycerol. Overnight cultures of freshly transformed E. coli strains are grown for 12-16 h in Terrific Broth (TB) at 37° C. and used to inoculate TB (50 ml) with 1.5% (w/v) glycerol and appropriate antibiotics to an optical density at 600 nm (OD600) of 0.05 in a 250 ml-baffled flask. The cultures are grown at 37° C. in a rotary shaker (200 r.p.m.) and induced with IPTG (1.0 mM) and L-arabinose (0.2% (w/v) (these inducers are dependent on the promoters used for plasmid construction; choice of inducer are apparent to those of skill in the art) at OD600=0.35-0.45. At this time, the growth temperature is reduced to 30° C., and the culture flasks are sealed with butyl rubber stoppers to prevent propylene evaporation. Additional glucose (1% (w/v)) is added concurrent with culture sampling after 1 d. Flasks are unsealed for 10 to 30 min every 24 h then resealed after sampling. Samples are drawn from both the liquid phase and headspace using a 10 μL Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m×0.32 mm column and an FID maintained at 200° C. The injector is connected in splitless mode and maintained at 250° C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50° C. 1.5 min; ramp to 300° C. at 10° C./min; hold at 300° C. 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H₂O. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell.

Production of Propylene from glucose. Overnight cultures of freshly transformed E. coli strains are grown for 12-16 h in Terrific Broth (TB) at 37° C. and used to inoculate TB (50 ml) with 1.5% (w/v) glucose replacing the standard glycerol supplement and appropriate antibiotics to an optical density at 600 nm (OD600) of 0.05 in a 250 ml-baffled flask. The cultures are grown at 37° C. in a rotary shaker (200 r.p.m.) and induced with IPTG (1.0 mM) and L-arabinose (0.2% (w/v)) at OD600=0.35-0.45. At this time, the growth temperature is reduced to 30° C., and the culture flasks are sealed with butyl rubber stoppers to prevent propylene evaporation. Additional glucose (1% (w/v)) is added concurrent with culture sampling after 1 d. Flasks are unsealed for 10 to 30 min every 24 h then resealed after sampling. Samples are drawn from both the liquid phase and headspace using a 10 μL Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m×0.32 mm column and an FID maintained at 200° C. The injector is connected in splitless mode and maintained at 250° C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50° C. 1.5 min; ramp to 300° C. at 10° C./min; hold at 300° C. 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H₂O. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell.

The disclosure of U.S. Provisional Application No. 61/702,534, filed on Sep. 18, 2012, and U.S. Non-provisional Application No. 14/429,327, filed on Mar. 18, 2015, are incorporated herein by reference in their entirety.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including U.S. Provisional Application No. 61/702,534 filed on Sep. 18, 2012, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. 

What is claimed is:
 1. A modified methanotrophic bacterium, wherein the modified methanotrophic bacterium comprises: (a) a heterologous nucleic acid molecule encoding a crotonase, wherein the modified methanotrophic bacterium produces crotonyl-CoA from a C₁ substrate comprising methane; and (b) a parent methanotrophic bacterium selected from Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomonas methanica 16a, Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylobacterium organophilum, Methylomonas sp AJ-3670, Methylocella silvestris, Methylomicrobium alcaliphilum, or a growth variant thereof; and wherein the modified methanotrophic bacterium produces n-butanol, butyraldehyde, or 1,4-butanediol.
 2. The modified methanotrophic bacterium of claim 1, wherein the methanotrophic bacterium is an obligate methanotrophic bacterium.
 3. The modified methanotrophic bacterium of claim 1, wherein the heterologous nucleic acid molecule further encodes a butyryl-CoA dehydrogenase, whereby a butyryl-CoA intermediate is produced and the modified methanotrophic bacterium produces n-butanol, butyraldehyde, or both.
 4. The modified methanotrophic bacterium of claim 3, wherein the heterologous nucleic acid molecule further encodes an aldehyde dehydrogenase, and the modified methanotrophic bacterium produces n-butanol, butyraldehyde, or both.
 5. The modified methanotrophic bacterium of claim 1, wherein the heterologous nucleic acid molecule further encodes a crotonyl-CoA hydratase, whereby a hydroxybutyryl-CoA intermediate is produced and the modified methanotrophic bacterium produces 1,4-butanediol.
 6. The modified methanotrophic bacterium of claim 4, wherein the modified methanotrophic bacterium does not have a functional PHB synthase or a substantial amount of functional PHB synthase.
 7. The modified methanotrophic bacterium of claim 5, wherein the modified methanotrophic bacterium does not have a functional PHB synthase or a substantial amount of functional PHB synthase.
 8. The modified methanotrophic bacterium of claim 6, wherein the PHB synthase is encoded by phaC or phbC.
 9. The modified methanotrophic bacterium of claim 7, wherein the PHB synthase is encoded by phaC or phbC.
 10. The modified methanotrophic bacterium of claim 4, wherein the modified methanotrophic bacterium has a functional β-ketothiolase activity and a functional acetoacetyl coenzyme A reductase activity.
 11. The modified methanotrophic bacterium of claim 5, wherein the modified methanotrophic bacterium has a functional β-ketothiolase activity and a functional acetoacetyl coenzyme A reductase activity.
 12. The modified methanotrophic bacterium of claim 10, wherein the β-ketothiolase is encoded by phaA or phbA.
 13. The modified methanotrophic bacterium of claim 10, wherein the acetoacetyl coenzyme A reductase is encoded by phaB or phbB.
 14. The modified methanotrophic bacterium of claim 11, wherein the β-ketothiolase is encoded by phaA or phbA.
 15. The modified methanotrophic bacterium of claim 11, wherein the acetoacetyl coenzyme A reductase is encoded by phaB or phbB.
 16. The modified methanotrophic bacterium of claim 6, wherein the modified methanotrophic bacterium does not produce a substantial amount of polyhydroxybutyrate.
 17. The modified methanotrophic bacterium of claim 7, wherein the modified methanotrophic bacterium does not produce a substantial amount of polyhydroxybutyrate.
 18. The modified methanotrophic bacterium of claim 1, wherein the C₁ substrate comprising methane comprises natural gas.
 19. A method for producing n-butanol and/or butyraldehyde, comprising culturing the modified methanotrophic bacterium of claim 1 on a C1 substrate comprising methane under conditions sufficient to produce n-butanol and/or butyraldehyde.
 20. A method for producing 1,4-butanediol, comprising culturing the modified methanotrophic bacterium of claim 1 on a C1 substrate comprising methane under conditions sufficient to produce 1,4-butanediol. 